siRNA useful to supress expression of EIF-5A1

ABSTRACT

The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as apoptosis factor 5A1 or simply factor 5A1, apoptosis factor 5A1 nucleic acids and polypeptides and methods for inhibiting or suppressing apoptosis in cells using antisense nucleotides or siRNAs to inhibit expression of factor 5A1. The invention also relates to suppressing or inhibiting expression of pro-inflammatory cytokines by inhibiting expression of apoptosis factor 5A.

RELATED APPLICATIONS

This application is a divisional application of Ser. No. 11/287,460,filed on Nov. 28, 2005 now U.S. Pat. No. 7,662,796, which is acontinuation of U.S. application Ser. No. 11/078,526, filed on Mar. 14,2005 now abandoned, which is a continuation of U.S. application Ser. No.10/792,893, filed on Mar. 5, 2004 now abandoned, and also claimspriority to U.S. provisional 60/476,194, filed on Jun. 6, 2003; and U.S.provisional 60/504,731, filed on Sep. 22, 2003, all of which are hereinincorporated in their entirety.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled“061945-5008-03-SequenceListing.txt” created on or about 3 Nov. 2011with a file size of about 46 kb contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to apoptosis-specific eucaryoticinitiation Factor-5A (eIF-5A) or referred to as apoptosis Factor 5A orFactor 5A1 and deoxyhypusine synthase (DHS). The present inventionrelates to apoptosis Factor 5A and DHS nucleic acids and polypeptidesand methods for inhibiting expression of apoptosis Factor 5A and DHS.

BACKGROUND OF THE INVENTION

Apoptosis is a genetically programmed cellular event that ischaracterized by well-defined morphological features, such as cellshrinkage, chromatin condensation, nuclear fragmentation, and membraneblebbing. Kerr et al. (1972) Br. J. Cancer, 26, 239-257; Wyllie et al.(1980) Int. Rev. Cytol., 68, 251-306. It plays an important role innormal tissue development and homeostasis, and defects in the apoptoticprogram are thought to contribute to a wide range of human disordersranging from neurodegenerative and autoimmunity disorders to neoplasms.Thompson (1995) Science, 267, 1456-1462; Mullauer et al. (2001) Mutat.Res, 488, 211-231. Although the morphological characteristics ofapoptotic cells are well characterized, the molecular pathways thatregulate this process have only begun to be elucidated.

One group of proteins that is thought to play a key role in apoptosis isa family of cysteine proteases, termed caspases, which appear to berequired for most pathways of apoptosis. Creagh & Martin (2001) Biochem.Soc. Trans, 29, 696-701; Dales et al. (2001) Leuk. Lymphoma, 41,247-253. Caspases trigger apoptosis in response to apoptotic stimuli bycleaving various cellular proteins, which results in classicmanifestations of apoptosis, including cell shrinkage, membrane blebbingand DNA fragmentation. Chang & Yang (2000) Microbiol. Mol. Biol. Rev.,64, 821-846.

Pro-apoptotic proteins, such as Bax or Bak, also play a key role in theapoptotic pathway by releasing caspase-activating molecules, such asmitochondrial cytochrome c, thereby promoting cell death throughapoptosis. Martinou & Green (2001) Nat. Rev. Mol. Cell. Biol., 2, 63-67;Zou et al. (1997) Cell, 90, 405-413. Anti-apoptotic proteins, such asBcl-2, promote cell survival by antagonizing the activity of thepro-apoptotic proteins, Bax and Bak. Tsujimoto (1998) Genes Cells, 3,697-707; Kroemer (1997) Nature Med., 3, 614-620. The ratio of Bax:Bcl-2is thought to be one way in which cell fate is determined; an excess ofBax promotes apoptosis and an excess of Bcl-2 promotes cell survival.Salomons et al. (1997) Int. J. Cancer, 71, 959-965; Wallace-Brodeur &Lowe (1999) Cell Mol. Life. Sci., 55, 64-75.

Another key protein involved in apoptosis is that encoded by the tumorsuppressor gene p53. This protein is a transcription factor thatregulates cell growth and induces apoptosis in cells that are damagedand genetically unstable, presumably through up-regulation of Bax. Boldet al. (1997) Surgical Oncology, 6, 133-142; Ronen et al., 1996; Schuler& Green (2001) Biochem. Soc. Trans., 29, 684-688; Ryan et al. (2001)Curr. Opin. Cell Biol., 13, 332-337; Zörnig et al. (2001) Biochem.Biophys. Acta, 1551, F1-F37.

The distinct morphological features that characterize cells undergoingapoptosis have given rise to a number of methods for assessing the onsetand progress of apoptosis. One such feature of apoptotic cells that canbe exploited for their detection is activation of a flippase, whichresults in externalization of phosphatidylserine, a phospholipidnormally localized to the inner leaflet of the plasma membrane. Fadok etal. (1992) J. Immunol., 149, 4029-4035. Apoptotic cells bearingexternalized phosphatidylserine can be detected by staining with aphosphatidylserine-binding protein, Annexin V, conjugated to afluorescent dye. The characteristic DNA fragmentation that occurs duringthe apoptotic process can be detected by labeling the exposed 3′-OH endsof the DNA fragments with fluorescein-labeled deoxynucleotides.Fluorescent dyes that bind nucleic acids, such as Hoescht 33258, can beused to detect chromatin condensation and nuclear fragmentation inapoptotic cells. The degree of apoptosis in a cell population can alsobe inferred from the extent of caspase proteolytic activity present incellular extracts.

As a genetically defined process, apoptosis, like any otherdevelopmental program, can be disrupted by mutation. Alterations in theapoptotic pathways are believed to play a key role in a number ofdisease processes, including cancer. Wyllie et al. (1980) Int. Rev.Cytol., 68, 251-306; Thompson (1995) Science, 267, 1456-1462; Sen &D'Incalci (1992) FEBS Letters, 307, 122-127; McDonnell et al. (1995)Seminars in Cancer and Biology, 6, 53-60. Investigations into cancerdevelopment and progression have traditionally been focused on cellularproliferation. However, the important role that apoptosis plays intumorigenesis has recently become apparent. In fact, much of what is nowknown about apoptosis has been learned using tumor models, since thecontrol of apoptosis is invariably altered in some way in tumor cells.Bold et al. (1997) Surgical Oncology, 6, 133-142.

Apoptosis can be triggered during tumor development by a variety ofsignals. Extracellular signals include growth or survival factordepletion, hypoxia and ionizing radiation. Internal signals that cantrigger apoptosis include DNA damage, shortening telomeres, andoncogenic mutations that produce inappropriate proliferative signals.Lowe & Lin (2000) Carcinogenesis, 21, 485-495. Ionizing radiation andnearly all cytotoxic chemotherapy agents used to treat malignancies arethought to act by triggering endogenous apoptotic mechanisms to inducecell death. Rowan & Fisher (1997) Leukemia, 11, 457-465; Kerr et al.(1994) Cancer, 73, 2013-2026; Martin & Schwartz (1997) OncologyResearch, 9, 1-5.

Evidence would suggest that early in the progression of cancer, tumorcells are sensitive to agents (such as ionizing radiation orchemotherapeutic drugs) that induce apoptosis. However, as the tumorprogresses, the cells develop resistance to apoptotic stimuli. Naik etal. (1996) Genes and Development, 10, 2105-2116. This may explain whyearly cancers respond better to treatment than more advanced lesions.The ability of late-stage cancers to develop resistance to chemotherapyand radiation therapy appears to be linked to alterations in theapoptotic pathway that limit the ability of tumor cells to respond toapoptotic stimuli. Reed et al. (1996) Journal of Cellular Biology, 60,23-32; Meyn et al. (1996) Cancer Metastasis Reviews, 15, 119-131; Hannun(1997) Blood, 89, 1845-1853; Reed (1995) Toxicology Letters, 82-83,155-158; Hickman (1996) European Journal of Cancer, 32A, 921-926.Resistance to chemotherapy has been correlated to overexpression of theanti-apoptotic gene bcl-2 and deletion or mutation of the pro-apoptoticbax gene in chronic lymphocytic leukemia and colon cancer, respectively.

The ability of tumor cells to successfully establish disseminatedmetastases also appears to involve alterations in the apoptotic pathway.Bold et al. (1997) Surgical Oncology, 6, 133-142. For example, mutationsin the tumor suppressor gene p53 are thought to occur in 70% of tumors.Evan et al. (1995) Curr. Opin. Cell Biol., 7, 825-834. Mutations thatinactivate p53 limit the ability of cells to induce apoptosis inresponse to DNA damage, leaving the cell vulnerable to furthermutations. Ko & Prives (1996) Genes and Development, 10, 1054-1072.

Therefore, apoptosis is intimately involved in the development andprogression of neoplastic transformation and metastases, and a betterunderstanding of the apoptotic pathways involved may lead to newpotential targets for the treatment of cancer by the modulation ofapoptotic pathways through gene therapy approaches. Bold et al. (1997)Surgical Oncology, 6, 133-142.

The present invention relates to cloning of an eIF-5A cDNA that is upregulated immediately before the induction of apoptosis. Thisapoptosis-specific eIF-5A is likely to be a suitable target forintervention in apoptosis-causing disease states since it appears to actat the level of post-transcriptional regulation of downstream effectorsand transcription factors involved in the apoptotic pathway.Specifically, the apoptosis-specific eIF-5A appears to selectivelyfacilitate the translocation of mRNAs encoding downstream effectors andtranscription factors of apoptosis from the nucleus to the cytoplasm,where they are subsequently translated. The ultimate decision toinitiate apoptosis appears to stem from a complex interaction betweeninternal and external pro- and anti-apoptotic signals. Lowe & Lin (2000)Carcinogenesis, 21, 485-495. Through its ability to facilitate thetranslation of downstream apoptosis effectors and transcription factors,the apoptosis-related eIF-5A appears to tip the balance between thesesignals in favor of apoptosis.

As described previously, it is well established that anticancer agentsinduce apoptosis and that alterations in the apoptotic pathways canattenuate drug-induced cell death. Schmitt & Lowe (1999) J. Pathol.,187, 127-137. For example, many anticancer drugs upregulate p53, andtumor cells that have lost p53 develop resistance to these drugs.However, nearly all chemotherapy agents can induce apoptosisindependently of p53 if the dose is sufficient, indicating that even indrug-resistant tumors, the pathways to apoptosis are not completelyblocked. Wallace-Brodeur & Lowe (1999) Cell Mol. Life. Sci., 55, 64-75.This suggests that induction of apoptosis eIF-5A, even though it may notcorrect the mutated gene, may be able to circumvent the p53-dependentpathway and induce apoptosis by promoting alternative pathways.

Induction of apoptosis-related eIF-5A has the potential to selectivelytarget cancer cells while having little or no effect on normalneighboring cells. This arises because mitogenic oncogenes expressed intumor cells provide an apoptotic signal in the form of specific speciesof mRNA that are not present in normal cells. Lowe et al. (1993) Cell,74, 954-967; Lowe & Lin (2000) Carcinogenesis, 21, 485-495. For example,restoration of wild-type p53 in p53-mutant tumor cells can directlyinduce apoptosis as well as increase drug sensitivity in tumor celllines and xenographs. (Spitz et al., 1996; Badie et al. 1998).

The selectivity of apoptosis-eIF-5A arises from the fact that itselectively facilitates translation of mRNAs for downstream apoptosiseffectors and transcription factors by mediating their translocationfrom the nucleus into the cytoplasm. Thus, for apoptosis eIF-5A to havean effect, mRNAs for these effectors and transcription factors have tobe transcribed. Inasmuch as these mRNAs would be transcribed in cancercells, but not in neighboring normal cells, it is to be expected thatapoptosis eIF-5A would promote apoptosis in cancer cells but haveminimal, if any, effect on normal cells. Thus, restoration of apoptoticpotential in tumor cells with apoptosis-related eIF-5A may decrease thetoxicity and side effects experienced by cancer patients due toselective targeting of tumor cells. Induction of apoptotic eIF-5A alsohas the potential to potentiate the response of tumor cells toanti-cancer drugs and thereby improve the effectiveness of these agentsagainst drug-resistant tumors. This in turn could result in lower dosesof anti-cancer drugs for efficacy and reduced toxicity to the patient.

Alternations in the apoptotic pathways are also believed to play a rolein degeneration of retinal ganglion cells causing blindness due toglaucoma. Glaucoma describes a group of eye conditions in whichincreased intra-ocular pressure (IOP) leads to damage of the optic nerveand progressive blindness. Although glaucoma is currently managed bydrugs or surgery to control IOP to reduce damage to the optic nerve orthe use of neuro-protectors, there remains a need to protect retinalganglion cells from degeneration by apoptosis in the glaucomatous eye.

Cytokines have also been implicated in the apoptotic pathway. Biologicalsystems require cellular interactions for their regulation, andcross-talk between cells generally involves a large variety ofcytokines. Cytokines are mediators that are produced in response to awide variety of stimuli by many different cell types. Cytokines arepleiotropic molecules that can exert many different effects on manydifferent cell types, but are especially important in regulation of theimmune response and hematopoietic cell proliferation anddifferentiation. The actions of cytokines on target cells can promotecell survival, proliferation, activation, differentiation, or apoptosisdepending on the particular cytokine, relative concentration, andpresence of other mediators.

The use of anti-cytokines to treat autoimmune disorders (psoriasis,rheumatoid arthritis, Crohn's disease) is gaining popularity. Thepro-inflammatory cytokines IL-1 and TNF play a large role in thepathology of these chronic disorders and anti-cytokine to therapies thatreduce the biological activities of these two cytokines can providetherapeutic benefits (Dinarello and Abraham, 2002).

Interleukin 1 (IL-1) is an important cytokine that mediates local andsystemic inflammatory reactions and which can synergize with TNF in thepathogenesis of many disorders, including vasculitis, osteoporosis,neurodegenerative disorders, diabetes, lupus nephritis, and autoimmunedisorders such as rheumatoid arthritis. The importance of IL-1β intumour angiogenesis and invasiveness was also recently demonstrated bythe resistance of IL-1β knockout mice to metastases and angiogenesiswhen injected with melanoma cells (Voronov et al., 2003).

Interleukin 18 (IL-18) is a recently discovered member of the IL-1family and is related by structure, receptors, and function to IL-1.IL-18 is a central cytokine involved in inflammatory and autoimmunedisorders as a result of its ability to induce interferon-gamma (IFN-λ),TNF-α, and IL-1. IL-1β and IL-18 are both capable of inducing productionof TNF-α, a cytokine known to contribute to cardiac dysfunction duringmyocardial ischemia (Maekawa et al., 2002). Inhibition of IL-18 byneutralization with an IL-18 binding protein was found to reduceischemia-induced myocardial dysfunction in an ischemia/reperfusion modelof suprafused human atrial myocardium (Dinarello, 2001). Neutralizationof IL-18 using a mouse IL-18 binding protein was also able to decreaseIFN-λ, TNF-α, and IL-1β transcript levels and reduce joint damage in acollagen-induced arthritis mouse model (Banda et al., 2003). A reductionof IL-18 production or availability may also prove beneficial to controlmetastatic cancer as injection of IL-18 binding protein in a mousemelanoma model successfully inhibited metastases (Carrascal et al.,2003). As a further indication of its importance as a pro-inflammatorycytokine, plasma levels of IL-18 were elevated in patients with chronicliver disease and increased levels were correlated with the severity ofthe disease (Ludwiczek et al., 2002). Similarly, IL-18 and TNF-α wereelevated in the serum of diabetes mellitus patients with nephropathy(Moriwaki et al., 2003). Neuroinflammation following traumatic braininjury is also mediated by pro-inflammatory cytokines and inhibition ofIL-18 by the IL-18 binding protein improved neurological recovery inmice following brain trauma (Yatsiv et al., 2002).

TNF-α, a member of the TNF family of cytokines, is a pro-inflammatorycytokine with pleiotropic effects ranging from co-mitogenic effects onhematopoietic cells, induction of inflammatory responses, and inductionof cell death in many cell types. TNF-α is normally induced by bacteriallipopolysaccharides, parasites, viruses, malignant cells and cytokinesand usually acts beneficially to protect cells from infection andcancer. However, inappropriate induction of TNF-α is a major contributorto disorders resulting from acute and chronic inflammation such asautoimmune disorders and can also contribute to cancer, AIDS, heartdisease, and sepsis (reviewed by Aggarwal and Natarajan, 1996; Sharmaand Anker, 2002). Experimental animal models of disease (i.e. septicshock and rheumatoid arthritis) as well as human disorders (i.e.inflammatory bowel diseases and acute graft-versus-host disease) havedemonstrated the beneficial effects of blocking TNF-α (Wallach et al.,1999). Inhibition of TNF-α has also been effective in providing reliefto patients suffering autoimmune disorders such as Crohn's disease (vanDeventer, 1999) and rheumatoid arthritis (Richard-Miceli and Dougados,2001). The ability of TNF-α to promote the survival and growth of Blymphocytes is also thought to play a role in the pathogenesis of B-cellchronic lymphocytic leukemia (B-CLL) and the levels of TNF-α beingexpressed by T cells in B-CLL was positively correlated with tumour massand stage of the disease (Bojarska-Junak et al., 2002). Interleukin-1β(IL-1β) is a cytokine known to induce TNF-α production.

Deoxyhypusine synthase (DHS) and hypusine-containing eucaryotictranslation initiation Factor-5A (eIF-5A) are known to play importantroles in many cellular processes including cell growth anddifferentiation. Hypusine, a unique amino acid, is found in all examinedeucaryotes and archaebacteria, but not in eubacteria, and eIF-5A is theonly known hypusine-containing protein. Park (1988) J. Biol. Chem., 263,7447-7449; Schümann & Klink (1989) System. Appl. Microbiol., 11,103-107; Bartig et al. (1990) System. Appl. Microbiol., 13, 112-116;Gordon et al. (1987a) J. Biol. Chem., 262, 16585-16589. Active eIF-5A isformed in two post-translational steps: the first step is the formationof a deoxyhypusine residue by the transfer of the 4-aminobutyl moiety ofspermidine to the α-amino group of a specific lysine of the precursoreIF-5A catalyzed by deoxyhypusine synthase; the second step involves thehydroxylation of this 4-aminobutyl moiety by deoxyhypusine hydroxylaseto form hypusine.

The amino acid sequence of eIF-5A is well conserved between species, andthere is strict conservation of the amino acid sequence surrounding thehypusine residue in eIf-5A, which suggests that this modification may beimportant for survival. Park et al. (1993) Biofactors, 4, 95-104. Thisassumption is further supported by the observation that inactivation ofboth isoforms of eIF-5A found to date in yeast, or inactivation of theDHS gene, which catalyzes the first step in their activation, blockscell division. Schnier et al. (1991) Mol. Cell. Biol., 11, 3105-3114;Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J.Biol. Chem., 273, 1677-1683. However, depletion of eIF-5A protein inyeast resulted in only a small decrease in total protein synthesissuggesting that eIF-5A may be required for the translation of specificsubsets of mRNA's rather than for protein global synthesis. Kang et al.(1993), “Effect of initiation factor eIF-5A depletion on cellproliferation and protein synthesis,” in Tuite, M. (ed.), ProteinSynthesis and Targeting in Yeast, NATO Series H. The recent finding thatligands that bind eIF-5A share highly conserved motifs also supports theimportance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276, 2555-2561.In addition, the hypusine residue of modified eIF-5A was found to beessential for sequence-specific binding to RNA, and binding did notprovide protection from ribonucleases.

In addition, intracellular depletion of eIF-5A results in a significantaccumulation of specific mRNAs in the nucleus, indicating that eIF-5Amay be responsible for shuttling specific classes of mRNAs from thenucleus to the cytoplasm. Liu & Tartakoff (1997) Supplement to MolecularBiology of the Cell, 8, 426a. Abstract No. 2476, 37th American Societyfor Cell Biology Annual Meeting. The accumulation of eIF-5A at nuclearpore-associated intranuclear filaments and its interaction with ageneral nuclear export receptor further suggest that eIF-5A is anucleocytoplasmic shuttle protein, rather than a component of polysomes.Rosorius et al. (1999) J. Cell Science, 112, 2369-2380.

The first cDNA for eIF-5A was cloned from human in 1989 by Smit-McBrideet al., and since then cDNAs or genes for eIF-5A have been cloned fromvarious eukaryotes including yeast, rat, chick embryo, alfalfa, andtomato. Smit-McBride et al. (1989a) J. Biol. Chem., 264, 1578-1583;Schnier et al. (1991) (yeast); Sano, A. (1995) in Imahori, M. et al.(eds), Polyamines, Basic and Clinical Aspects, VNU Science Press, TheNetherlands, 81-88 (rat); Rinaudo & Park (1992) FASEB J., 6, A453 (chickembryo); Pay et al. (1991) Plant Mol. Biol., 17, 927-929 (alfalfa); Wanget al. (2001) J. Biol. Chem., 276, 17541-17549 (tomato).

Expression of eIF-5A mRNA has been explored in various human tissues andmammalian cell lines. For example, changes in eIF-5A expression havebeen observed in human fibroblast cells after addition of serumfollowing serum deprivation. Pang & Chen (1994) J. Cell Physiol., 160,531-538. Age-related decreases in deoxyhypusine synthase activity andabundance of precursor eIF-5A have also been observed in senescingfibroblast cells, although the possibility that this reflects averagingof differential changes in isoforms was not determined. Chen & Chen(1997b) J. Cell Physiol., 170, 248-254.

Studies have shown that eIF-5A may be the cellular target of viralproteins such as the human immunodeficiency virus type 1 Rev protein andhuman T cell leukemia virus type 1 Rex protein. Ruhl et al. (1993) J.Cell Biol., 123, 1309-1320; Katahira et al. (1995) J. Virol., 69,3125-3133. Preliminary studies indicate that eIF-5A may target RNA byinteracting with other RNA-binding proteins such as Rev, suggesting thatthese viral proteins may recruit eIF-5A for viral RNA processing. Liu etal. (1997) Biol. Signals, 6, 166-174.

Thus, although eIF-5A and DHS are known, there remains a need inunderstanding how these proteins are involved in apoptotic pathways aswell as cytokine stimulation to be able to modulate apoptosis andcytokine expression. The present invention fulfills this need.

SUMMARY OF INVENTION

The present invention relates to apoptosis specific eucaryoticinitiation factor 5A (eIF-5A), referred to as apoptosis factor 5A1 orsimply factor 5A1. The present invention also relates to apoptosisfactor 5A1 nucleic acids and polypeptides and methods for inhibiting orsuppressing apoptosis in cells using antisense nucleotides or siRNAs toinhibit expression of factor 5A 1. The invention also relates tosuppressing or inhibiting expression of pro-inflammatory cytokines byinhibiting expression of apoptosis factor 5A1. Further, the presentinvention relates to inhibiting or suppressing expression of p53 byinhibiting expression of apoptosis factor 5A1. The present inventionalso relates to a method of increasing Bcl-2 expression by inhibiting orsuppressing expression of apoptosis factor 5A1 using antisensenucleotides or siRNAs. The present invention also provides a method ofinhibiting production of cytokines, especially TNF-α in human epithelialcells. In another embodiment of the present invention, suppressingexpression of apoptosis-specific eIF-5A1 by the use of antisenseoligonucleotides targeted at apoptosis-specific eIF-5A1 provides methodsof preventing retinal ganglion cell death in a glaucomatous eye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID NO: 11) and derived aminoacid sequence (SEQ ID NO: 12) of the 3′ end of rat apoptosis-specificeIF-5A.

FIG. 2 depicts the nucleotide sequence (SEQ ID NO: 15) and derived aminoacid sequence (SEQ ID NO: 16) of the 5′ end of rat apoptosis-specificeIF-5A cDNA.

FIG. 3 depicts the nucleotide sequence of rat corpus luteumapoptosis-specific eIF-5A full-length cDNA (SEQ ID NO: 1). The aminoacid sequence is shown in SEQ ID NO: 2.

FIG. 4 depicts the nucleotide sequence (SEQ ID NO: 6) and derived aminoacid sequence (SEQ ID NO: 7) of the 3′ end of rat apoptosis-specific DHScDNA.

FIG. 5 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) with thenucleotide sequence of human eIF-5A (SEQ ID NO: 3) (Accession numberBC000751 or NM_(—)001970, SEQ ID NO:3).

FIG. 6 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific cDNA (SEQ ID NO: 20) with thenucleotide sequence of human eIF-5A (SEQ ID NO: 4) (Accession numberNM_(—)020390, SEQ ID NO:4).

FIG. 7 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) with thenucleotide sequence of mouse eIF-5A (Accession number BC003889). Mousenucleotide sequence (Accession number BC003889) is SEQ ID NO:5.

FIG. 8 is an alignment of the derived full-length amino acid sequence ofrat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO: 2) with thederived amino acid sequence of human eIF-5A (SEQ ID NO: 21) (Accessionnumber BC000751 or NM_(—)001970).

FIG. 9 is an alignment of the derived full-length amino acid sequence ofrat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO: 2) with thederived amino acid sequence to of human eIF-5A (SEQ ID NO: 22)(Accession number NM_(—)020390).

FIG. 10 is an alignment of the derived full-length amino acid sequenceof rat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO: 2) with thederived amino acid sequence of mouse eIF-5A (SEQ ID NO: 23) (Accessionnumber BC003889).

FIG. 11 is an alignment of the partial-length nucleotide sequence of ratcorpus luteum apoptosis-specific DHS cDNA (residues 1-453 of SEQ ID NO:6) with the nucleotide sequence of human DHS (SEQ ID NO: 8) (Accessionnumber BC000333, SEQ ID NO:8).

FIG. 12 is a restriction map of rat corpus luteum apoptosis-specificeIF-5A cDNA.

FIG. 13 is a restriction map of the partial-length ratapoptosis-specific DHS cDNA.

FIG. 14 is a Northern blot (top) and an ethidium bromide stained gel(bottom) of total RNA probed with the ³²P-dCTP-labeled 3′-end of ratcorpus luteum apoptosis-specific eIF-5A cDNA.

FIG. 15 is a Northern blot (top) and an ethidium bromide stained gel(bottom) of total RNA probed with the ³²P-dCTP-labeled 3′-end of ratcorpus luteum apoptosis-specific DHS cDNA.

FIG. 16 depicts a DNA laddering experiment in which the degree ofapoptosis in superovulated rat corpus lutea was examined after injectionwith PGF-2α.

FIG. 17 is an agarose gel of genomic DNA isolated from apoptosing ratcorpus luteum showing DNA laddering after treatment of rats with PGFF-2α.

FIG. 18 depicts a DNA laddering experiment in which the degree ofapoptosis in dispersed cells of superovulated rat corpora lutea wasexamined in rats treated with spermidine prior to exposure toprostaglandin F-2α (PGF-2α).

FIG. 19 depicts a DNA laddering experiment in which the degree ofapoptosis in superovulated rat corpus lutea was examined in rats treatedwith spermidine and/or PGF-2α.

FIG. 20 is a Southern blot of rat genomic DNA probed with³²P-dCTP-labeled partial-length rat corpus luteum apoptosis-specificeIF-5A cDNA.

FIG. 21 depicts pHM6, a mammalian epitope tag expression vector (RocheMolecular Biochemicals).

FIG. 22 is a Northern blot (top) and ethidium bromide stained gel(bottom) of total RNA isolated from COS-7 cells after induction ofapoptosis by withdrawal of serum probed with the ³²P-dCTP-labeled3′-untranslated region of rat corpus luteum apoptosis-specific DHS cDNA.

FIG. 23 is a flow chart illustrating the procedure for transienttransfection of COS-7 cells.

FIG. 24 is a Western blot of transient expression of foreign proteins inCOS-7 cells following transfection with pHM6.

FIG. 25 illustrates enhanced apoptosis as reflected by increased caspaseactivity when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 26 illustrates enhanced apoptosis as reflected by increased DNAfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 27 illustrates detection of apoptosis as reflected by increasednuclear fragmentation when COS-7 cells were transiently transfected withpHM6 containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 28 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 29 illustrates detection of apoptosis as reflected byphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 30 illustrates enhanced apoptosis as reflected by increasedphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 31 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 32 illustrates enhanced apoptosis when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 33 illustrates down-regulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. The top photo is theCoomassie-blue-stained protein blot; the bottom photo is thecorresponding Western blot.

FIG. 34 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of COS-7 cells transiently transfected with pHM6 containingfull-length rat apoptosis-specific eIF-5A in the antisense orientationusing Bcl-2 as a probe.

FIG. 35 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of COS-7 cells transiently transfected with pHM6 containingfull-length rat apoptosis-specific eIF-5A in the sense orientation usingc-Myc as a probe.

FIG. 36 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of COS-7 cells transiently transfected with pHM6 containingfull-length rat apoptosis-specific eIF-5A in the sense orientation whenp53 is used as a probe.

FIG. 37 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of expression of pHM6-full-length rat apoptosis-specificeIF-5A in COS-7 cells using an anti-[HA]-peroxidase probe and aCoomassie-blue-stained protein blot and the corresponding Western blotof expression of pHM6-full-length rat apoptosis-specific eIF-5A in COS-7cells when a p53 probe is used.

FIG. 38 is an alignment of human eIF-5A2 isolated from RKO cells (SEQ IDNO: 24) with the sequence of human eIF-5A2 (SEQ ID NO: 22) (Genbankaccession number XM_(—)113401). The consensus sequence is shown in SEQID NO: 28.

FIG. 39 is a graph depicting the percentage of apoptosis occurring inRKO and RKO-E6 cells following transient transfection. RKO and RKO-E6cells were transiently transfected with pHM6-LacZ or pHM6-eIF-5A1. RKOcells treated with Actinomycin D and transfected with pHM6-eIF-5A1showed a 240% increase in apoptosis relative to cells transfected withpHM6-LacZ that were not treated with Actinomycin D. RKO-E6 cells treatedwith Actinomycin D and transfected with pHM6-eIF-5A1 showed a 105%increase in apoptosis relative to cells transfected with pHM6-LacZ thatwere not treated with Actinomycin D.

FIG. 40 is a graph depicting the percentage of apoptosis occurring inRKO cells following transient transfection. RKO cells were transientlytransfected with pHM6-LacZ, pHM6-eIF-5A1, pHM6-eIF-5A2, orpHM6-truncated eIF-5A1. Cells transfected with pHM6-eIF-5A1 showed a 25%increase in apoptosis relative to control cells transfected withpHM6-LacZ. This increase was not apparent for cells transfected withpHM6-eIF-5A2 or pHM6-truncated eIF-5A1.

FIG. 41 is a graph depicting the percentage of apoptosis occurring inRKO cells following transient transfection. RKO cells were either leftuntransfected or were transiently transfected with pHM6-LacZ orpHM6-eIF-5A1. After correction for transfection efficiency, 60% of thecells transfected with pHM6-eIF-5A1 were apoptotic.

FIG. 42 provides the results of a flow cytometry analysis of RKO cellapoptosis following transient transfection. RKO cells were either leftuntransfected or were transiently transfected with pHM6-LacZ,pHM6-eIF-5A1, pHM6-eIF-5A2, or pHM6-truncated eIF-5A1. The table depictsthe percentage of cells undergoing apoptosis calculated based on thearea under the peak of each gate. After correction for backgroundapoptosis in untransfected cells and for transfection efficiency, 80% ofcells transfected with pHM6-eIF-5A1 exhibited apoptosis. Cellstransfected with pHM6-LacZ, pHM6-eIF-5A2 or pHM6-truncated eIF-5A1exhibited only background levels of apoptosis.

FIG. 43 provides Western blots of protein extracted from RKO cellstreated with 0.25 μg/ml Actinomycin D for 0, 3, 7, 24, and 48 hours. Thetop panel depicts a Western blot using anti-p53 as the primary antibody.The middle panel depicts a Western blot using anti-eIF-5A1 as theprimary antibody. The bottom panel depicts the membrane used for theanti-eIF-5A1 blot stained with Coomassie blue following chemiluminescentdetection to demonstrate equal loading. p53 and eIF-5A1 are bothupregulated by treatment with Actinomycin D.

FIG. 44 is a bar graph showing that both apoptosis-specific eIF-5A(eIF-5A) and proliferation eIF-5A (eIF5b) are expressed in heart tissue.The heart tissue was taken from patients receiving coronary arterybypass grafts (CABG). Gene expression levels of eIF-5A (light gray bar)are compared to eIF5b (dark gray bar). The X-axis are patient identifiernumbers. The Y-axis is pg/ng of 18s (picograms of message RNA overnanograms of ribosomal RNA 18S).

FIG. 45 is a bar graph showing that both apoptosis-specific (eIF5a) andproliferation eIF-5A (eIF5b) are expressed in heart tissue. The hearttissue was taken from patients receiving valve replacements. Geneexpression levels of eIF5a (light gray bar) are compared to eIF5b (darkgray bar). The X-axis are patient identifier numbers. The Y-axis ispg/ng of 18s (picograms of message RNA over nanograms of ribosomal RNA18S).

FIG. 46 is a bar graph showing the gene expression levels measured byreal-time PCR of apoptosis-specific (eIf5a) versus proliferation eIF-5A(eIF5b) in pre-ischemia heart tissue and post ischemia heart tissue. TheY-axis is pg/ng of 18s (picograms of message RNA over nanograms ofribosomal RNA 18S).

FIG. 47 depicts schematically an experiment performed on heart tissue.The heart tissue was exposed to normal oxygen levels and the expressionlevels of apoptosis-specific eIF-5A (eIF5a) and proliferating eIF-5A(eIF5b) measured. Later, the amount of oxygen delivered to the hearttissue was lowered, thus inducing hypoxia and ischemia, and ultimately,a heart attack in the heart tissue. The expression levels ofapoptosis-specific eIF-5A (eIF5a) and proliferating eIF-5A (eIF5b) weremeasured and compared to the expression levels of the heart tissuebefore it was damaged by ischemia.

FIG. 48 shows EKGs of heart tissue before and after the ischemia wasinduced.

FIG. 49 shows the lab bench with the set up of the experiment depictedin FIG. 47.

FIGS. 50A-F report patient data where the levels of Apoptosis FactoreIF-5a (also denoted as eIf-5a1 or on the chart as IF5a1) are correlatedwith levels of IL-1β and IL-18. FIG. 50A is a chart of data obtainedfrom coronary artery bypass graft (CABG) patients. FIG. 50B is a chartof data obtained from valve replacement patients. FIG. 50C is a graphdepicting the correlation of apoptosis factor eIF-5a (Factor 5a1) toIL-18 in CABG patients. FIG. 50D is a graph depicting the correlation ofproliferating eIF-5a (Factor a2) to IL-18 in CABG patients. FIG. 50E isa graph depicting the correlation of apoptosis factor eIF-5a (Factor5a1) to IL-18 in valve replacement patients. FIG. 50F is a graphdepicting the correlation of proliferating eIF-5a (Factor a2) to IL-18in valve replacement patients.

FIG. 51 is a chart of the patient's data from which patients data usedin FIGS. 50A-F was obtained.

FIG. 52 shows the levels of protein produced by RKO cells after beingtreated with antisense oligo 1, 2 and 3 (to apoptosis factor 5A). TheRKO cells produced less apoptosis factor 5A as well as less p53 afterhaving been transfected with the antisense apoptosis factor 5Anucleotides.

FIG. 53 shows uptake of the fluorescently labeled antisenseoligonucleotide.

FIGS. 54-58 show a decrease in the percentage of cells undergoingapoptosis in the cells having been treated with antisense apoptosisfactor 5A oligonucleotides as compared to cells not having beentransfected with the antisense apoptosis factor 5A oligonucleotides.

FIG. 59 shows that treating lamina cribrosa cells with TNF-α and/orcamptothecin caused an increase in the number of cells undergoingapoptosis.

FIGS. 60 and 61 show a decrease in the percentage of cells undergoingapoptosis in the cells having being treated with antisense apoptosisfactor 5A oligonucleotides as compared to cells not having beentransfected with the antisense apoptosis factor 5A oligonucleotides.

FIG. 62 shows that the lamina cribrosa cells uptake the labeled siRNAeither in the presence of serum or without serum.

FIG. 63 shows that cells transfected with apoptosis factor 5a siRNAproduced less apoptosis factor 5a protein and in addition, produced moreBcl-2 protein. A decrease in apoptosis factor 5A expression correlateswith an increase in BCL-2 expression.

FIG. 64 shows that cells transfected with apoptosis factor 5a siRNAproduced less apoptosis factor 5a protein.

FIGS. 65-67 show that cells transfected with apoptosis factor 5a siRNAhad a lower percentage of cells undergoing apoptosis after exposure tocamptothecin and TNF-α.

FIG. 68 are photographs of Hoescht-stained lamina cribrosa cell line#506 transfected with siRNA and treated with camptothecin and TNF-α fromthe experiment described in FIG. 67 and Example 13. The apoptosing cellsare seen as more brightly stained cells. They have smaller nucleibecause of chromatin condensing and are smaller and irregular in shape.

FIG. 69 shows that IL-1 exposed HepG2 cells transfected with apoptosisfactor 5A cells secreted less TNF-α than non-transfected cells.

FIG. 70 shows the sequence of human apoptosis factor 5a (SEQ ID NO:29)and the sequences of 5 siRNAs of the present invention (SEQ ID NO:30,31, 32, 33 and 34).

FIG. 71 shows the sequence of human apoptosis factor 5a (SEQ ID NO: 29)and the sequences of 3 antisense polynucleotides of the presentinvention (SEQ ID NOS: 63-65, respectively in order of appearance) aswell as the sequence of 3 target polynucleotides (SEQ ID NOS: 35-37,respectively in order of appearance).

FIG. 72 shows the binding position of three antisense oligonucleotides(SEQ ID NO:25-27, respectively in order of appearance) targeted againsthuman eIF-5A1. The full-length nucleotide sequence is SEQ ID NO: 19.

FIG. 73 a and b shows the nucleotide alignment (SEQ ID NO: 41 and 42,respectively in order of appearance) and amino acid alignment (SEQ IDNO: 43 and 22, respectively in order of appearance) of human eIF-5A1(apoptosis factor 5A) against human eIF-5A2 (proliferating eIF-5A).

FIG. 74A provides a picture of a Western blot where siRNAs againsteIF-5A1 have reduced if not inhibited the production of TNF-α intransfected HT-29 cells. FIG. 74B provides the results of an ELISA.

FIG. 75 provides the results of an ELISA. TNF-α production was reducedin cells treated with siRNAs against eIF-5A1 as compared to controlcells.

FIG. 76 shows the time course of the U-937 differentiation experiment.See Example 16.

FIG. 77 shows the results of a Western blot showing that eIF-5A1 isup-regulated during monocyte differentiation and subsequent TNF-αsecretion.

FIG. 78 depicts stem cell differentiation and the use of siRNAs againsteIF-5A1 to inhibit cytokine production.

FIG. 79 is a bar graph showing that IL-8 is produced in response toTNF-alpha as well as in response to interferon. This graph shows thatsiRNA against eIF-5A blocked almost all IL-8 produced in response tointerferon as well as a significant amount of the eIL-8 produced as aresult of the combined treatment of interferon and TNF.

FIG. 80 is another bar graph showing that IL-8 is produced in responseto TNF-alpha as well as in response to interferon. This graph shows thatsiRNA against eIF-5A blocked almost all IL-8 produced in response tointerferon as well as a significant amount of the eIL-8 produced as aresult of the combined treatment of interferon and TNF.

FIG. 81 is a western blot of HT-29 cells treated with IFN gamma for 8and 24 hours. This blot shows upregulation (4 fold at 8 hours) ofapoptosis eIF-5A in response to interferon gamma in HT-29 cells.

FIG. 82 is a characterization of lamina cribrosa cells byimmunofluorescence. Lamina cribrosa cells (#506) isolated from the opticnerve head of an 83-year old male were characterized byimmunofluorescence. Primary antibodies were a) actin; b) fibronectin; c)laminin; d) GFAP. All pictures were taken at 400 times magnification.

FIG. 83: Apoptosis of lamina cribrosa cell line #506 in response totreatment with camptothecin and TNF-α. Lamina cribrosa cell line #506cells were seeded at 40,000 cells per well onto an 8-well culture slide.Three days later the confluent LC cells were treated with either 10ng/ml TNF-α, 50 μM camptothecin, or 10 ng/ml TNF-α plus 50 camptothecin.An equivalent volume of DMSO, a vehicle control for camptothecin, wasadded to the untreated control cells. The cells were stained withHoescht 33258 48 hours after treatment and viewed by fluorescencemicroscopy using a UV filter. Cells with brightly stained condensed orfragmented nuclei were counted as apoptotic.

FIG. 84: Expression of eIF-5A during camptothecin or TNF-α pluscamptothecin treatment. Lamina cribrosa cell #506 cells were seeded at40,000 cells per well onto a 24-well plate. Three days later the LCcells were treated with either 50 μM camptothecin or 10 ng/ml TNF-α plus50 μM camptothecin and protein lysate was harvested 1, 4, 8, and 24hours later. An equivalent volume of DMSO was added to control cells asa vehicle control and cell lysate was harvested 1 and 24 hours later. 5μg of protein from each sample was separated by SDS-PAGE, transferred toa PVDF membrane, and Western blotted with anti-eIF-5A antibody. Thebound antibody was detected by chemiluminescence and exposed to x-rayfilm. The membrane was then stripped and re-blotted with anti-β-actin asan internal loading control.

FIG. 85: Expression of eIF-5A in lamina cribosa cell lines #506 and #517following transfection with siRNAs. Lamina cribrosa cell #506 and #517cells were seeded at 10,000 cells per well onto a 24-well plate. Threedays later the LC cells were transfected with either GAPDH siRNA, eIF-5AsiRNAs #1-4, or control siRNA #5. Three days after transfection theprotein lysate was harvested and 5 μg of protein from each sample wasseparated by SDS-PAGE, transferred to a PVDF membrane, and Westernblotted with anti-eIF-5A antibody. The bound antibody was detected bychemiluminescence and exposed to x-ray film. The membrane was thenstripped and re-blotted with anti-β-actin as an internal loadingcontrol.

FIG. 86: Apoptosis of lamina cribosa cell line #506 cells transfectedwith eIF-5A siRNAs and treated with TNF-α and camptothecin. Laminacribrosa cell line #506 cells were seeded at 7500 cells per well onto an8-well culture slide. Three days later the LC cells were transfectedwith either GAPDH siRNA, eIF-5A siRNAs #1-4, or control siRNA #5. 72hours after transfection, the transfected cells were treated with 10ng/ml TNF-α plus 50 μM camptothecin. Twenty-four hours later the cellswere stained with Hoescht 33258 and viewed by fluorescence microscopyusing a UV filter. Cells with brightly stained condensed or fragmentednuclei were counted as apoptotic. This graph represents the average ofn=4 independent experiments.

FIG. 87: Apoptosis of lamina cribosa cell line #517 cells transfectedwith eIF-5A siRNA #1 and treated with TNF-α and camptothecin. Laminacribrosa cell line #517 cells were seeded at 7500 cells per well onto an8-well culture slide. Three days later the LC cells were transfectedwith either eIF-5A siRNA #1 or control siRNA #5. 72 hours aftertransfection, the transfected cells were treated with 10 ng/ml TNF-αplus 50 μM camptothecin. Twenty-four hours later the cells were stainedwith Hoescht 33258 and viewed by fluorescence microscopy using a UVfilter. Cells with brightly stained condensed or fragmented nuclei werecounted as apoptotic. The results of two independent experiments arerepresented here.

FIG. 88: TUNEL-labeling of lamina cribosa cell line #506 cellstransfected with eIF-5A siRNA #1 and treated with TNF-α andcamptothecin. Lamina cribrosa cell line #506 cells were seeded at 7500cells per well onto an 8-well culture slide. Three days later the LCcells were transfected with either eIF-5A siRNA #1 or control siRNA #5.72 hours after transfection, the transfected cells were treated with 10ng/ml TNF-α plus 50 μM camptothecin. Twenty-four hours later the cellswere stained with Hoescht 33258 and DNA fragmentation was evaluated insitu using the terminal deoxynucleotidyl transferase-mediateddUTP-digoxigenin nick end labeling (TUNEL) method. Panel A representsthe slide observed by fluorescence microscopy using a fluorescein filterto visualize TUNEL-labeling of the fragmented DNA of apoptotic cells.Panel B represents the same slide observed through a UV filter tovisualize the Hoescht-stained nuclei. The results are representative oftwo independent experiments. All pictures were taken at 400 timesmagnification.

FIG. 89 depicts the design of siRNAs against eIF5-A1 (SEQ ID NO: 44-58,respectively in order of appearance). The siRNAs have the SEQ ID NO: 45,48, 51, 54 and 56. The full-length nucleotide sequence is show in SEQ IDNO: 29.

DETAILED DESCRIPTION OF THE INVENTION

Several isoforms of eukaryotic initiation factor 5a (eIF-5A) have beenisolated and present in published databanks. It was thought that theseisoforms were functionally redundant. The present inventors havediscovered that one isoform is upregulated immediately before theinduction of apoptosis, which they have designated apoptosis factor 5Aor factor 5A1 or eIF5-A1. The subject of the present invention isapoptosis factor 5A as well as DHS, which is involved in the activationof eIF-5A.

Apoptosis factor 5A is likely to be a suitable target for interventionin apoptosis-causing disease states since it appears to act at the levelof post-transcriptional regulation of downstream effectors andtranscription factors involved in the apoptotic pathway. Specifically,apoptosis factor 5A appears to selectively facilitate the translocationof mRNAs encoding downstream effectors and transcription factors ofapoptosis from the nucleus to the cytoplasm, where they are subsequentlytranslated. The ultimate decision to initiate apoptosis appears to stemfrom a complex interaction between internal and external pro- andanti-apoptotic signals. Lowe & Lin (2000) Carcinogenesis, 21, 485-495.Through its ability to facilitate the translation of downstreamapoptosis effectors and transcription factors, the apoptosis factor 5Aappears to tip the balance between these signals in favor of apoptosis.

Accordingly, the present invention provides a method of suppressing orreducing apoptosis in a cell by administering an agent that inhibits orreduces expression of either apoptosis factor 5A or DHS. One agent thatcan inhibit or reduce expression of apoptosis factor 5A or DHS is anantisense oligonucleotide.

Antisense oligonucleotides have been successfully used to accomplishboth in vitro as well as in vivo gene-specific suppression. Antisenseoligonucleotides are short, synthetic strands of DNA (or DNA analogs)that are antisense (or complimentary) to a specific DNA or RNA target.Antisense oligonucleotides are designed to block expression of theprotein encoded by the DNA or RNA target by binding to the target andhalting expression at the level of transcription, translation, orsplicing. By using modified backbones that resist degradation (Blake etal., 1985), such as replacement of the phosphodiester bonds in theoligonucleotides with phosphorothioate linkages to retard nucleasedegradation (Matzura and Eckstein, 1968), antisense oligonucleotideshave been used successfully both in cell cultures and animal models ofdisease (Hogrefe, 1999).

Preferably, the antisense oligonucleotides of the present invention havea nucleotide sequence encoding a portion of an apoptosis factor 5Apolypeptide or an apoptosis-specific DHS polypeptide. The inventors havetransfected various cell lines with antisense nucleotides encoding aportion of an apoptosis factor 5A polypeptide as described below andmeasured the number of cells undergoing apoptosis. The cells that weretransfected with the antisense oligonucleotides showed a decrease in thenumber of cells undergoing apoptosis as compared to like cells nothaving been transfected with the antisense oligos. FIGS. 54-58 show adecrease in the percentage of cells undergoing apoptosis in the cellshaving being treated with antisense apoptosis factor 5A oligonucleotidesas compared to cells not having been transfected with the antisenseapoptosis factor 5A oligonucleotides.

The present invention contemplates the use of many suitable nucleic acidsequences encoding an apoptosis factor 5A polypeptide or DHSpolypeptide. For example, SEQ ID NOS:1, 3, 4, 5, 11, 15, 19, 20, and 21(apoptosis-factor 5A nucleic acid sequences), SEQ ID NOS:6 and 8(apoptosis-specific DHS nucleic acid sequences), SEQ ID NOS:12 and 16(apoptosis factor 5A sequences), and SEQ ID NO:7 (apoptosis-specific DHSpolypeptide sequences), or portions thereof, provide suitable sequences.Other preferred apoptosis factor 5A antisense polynucleotide sequencesinclude SEQ ID NO: 63-65. Additional antisense nucleotides include thosethat have substantial sequence identity to those enumerated above (i.e.90% homology) or those having sequences that hybridize under highlystringent conditions to the enumerated SEQ ID NOs. Additionally, othersuitable sequences can be found using the known sequences as probesaccording to methods known in the art.

The antisense oligonucleotides of the present invention may be singlestranded, double stranded, DNA, RNA or a hybrid. The oligonucleotidesmay be modified by methods known in the art to increase stability,increase resistance to nuclease degradation or the like. Thesemodifications are known in the art and include, but are not limited tomodifying the backbone of the oligonucleotide, modifying the sugarmoieties, or modifying the base. Also inclusive in these modificationsare various DNA-RNA hybrids or constructs commonly referred to as“gapped” oligonucleotides.

The present invention provides other agents that can inhibit or reduceexpression of apoptosis factor 5A or DHS. One such agent is siRNAs.Small Inhibitory RNAs (siRNA) have been emerging as a viable alternativeto antisense oligonucleotides since lower concentrations are required toachieve levels of suppression that are equivalent or superior to thoseachieved with antisense oligonucleotides (Thompson, 2002). Longdouble-stranded RNAs have been used to silence the expression ofspecific genes in a variety of organisms such as plants, nematodes, andfruit flies. An RNase-III family enzyme called Dicer processes theselong double stranded RNAs into 21-23 nucleotide small interfering RNAswhich are then incorporated into an RNA-induced silencing complex(RISC). Unwinding of the siRNA activates RISC and allows thesingle-stranded siRNA to guide the complex to the endogenous mRNA bybase pairing. Recognition of the endogenous mRNA by RISC results in itscleavage and consequently makes it unavailable for translation.Introduction of long double stranded RNA into mammalian cells results ina potent antiviral response which can be bypassed by use of siRNAs.(Elbashir et al., 2001). SiRNA has been widely used in cell cultures androutinely achieves a reduction in specific gene expression of 90% ormore.

The use of siRNAs has also been gaining popularity in inhibiting geneexpression in animal models of disease. A recent study that demonstratedthat an siRNA against luciferase was able to block luciferase expressionfrom a co-transfected plasmid in a wide variety of organs in post-natalmice using a hydrodynamic injection delivery technique (Lewis et al.,2002). An siRNA against Fas, a receptor in the TNF family, injectedhydrodynamically into the tail vein of mice was able to transfectgreater than 80% of hepatocytes and decrease Fas expression in the liverby 90% for up to 10 days after the last injection (Song et al., 2003).The Fas siRNA was also able to protect mice from liver fibrosis andfulminant hepatitis. The development of sepsis in mice treated with alethal dose of lipopolysaccharide was inhibited by the use of an siRNAdirected against TNF a (Sørensen et al., 2003). SiRNA has the potentialto be a very potent drug for the inhibition of specific gene expressionin vivo in light of their long-lasting effectiveness in cell culturesand in vivo, their ability to transfect cells in vivo, and theirresistance to degradation in serum (Bertrand et al., 2002).

The present inventors have transfected cells with apoptosis factor 5AsiRNAs and studied the effects on expression of apoptosis factor 5A.FIG. 64 shows that cells transfected with apoptosis factor 5a siRNAproduced less apoptosis factor 5a protein. FIGS. 64-67 show that cellstransfected with apoptosis factor 5A siRNAs have a lower percentage ofcells undergoing apoptosis after exposure to camptothecin and TNF-α ascompared to cells not having been transfected with apoptosis factor 5AsiRNAs.

Preferred siRNAs include those that have SEQ ID NO: 30, 31, 32, 33, and34. Additional siRNAs include those that have substantial sequenceidentity to those enumerated (i.e. 90% homology) or those havingsequences that hybridize under highly stringent conditions to theenumerated SEQ ID NOs. FIG. 64 shows that cells transfected withapoptosis factor 5A siRNA produced less apoptosis factor 5A protein.

Many important human diseases are caused by abnormalities in the controlof apoptosis. These abnormalities can result in either a pathologicalincrease in cell number (e.g. cancer) or a damaging loss of cells (e.g.degenerative diseases). As non-limiting examples, the methods andcompositions of the present invention can be used to prevent or treatthe following apoptosis-associated diseases and disorders:neurological/neurodegenerative disorders (e.g., Alzheimer's,Parkinson's, Huntington's, Amyotrophic Lateral Sclerosis (Lou Gehrig'sDisease), autoimmune disorders (e.g., rheumatoid arthritis, systemiclupus erythematosus (SLE), multiple sclerosis), Duchenne MuscularDystrophy (DMD), motor neuron disorders, ischemia, heart ischemia,chronic heart failure, stroke, infantile spinal muscular atrophy,cardiac arrest, renal failure, atopic dermatitis, sepsis and septicshock, AIDS, hepatitis, glaucoma, diabetes (type 1 and type 2), asthma,retinitis pigmentosa, osteoporosis, xenograft rejection, and burninjury.

One such disease caused by abnormalities in the control of apoptosis isglaucoma. Apoptosis is a critical factor leading to blindness inglaucoma patients. Glaucoma is a group of eye conditions arising fromdamage to the optic nerve that results in progressive blindness.Apoptosis has been shown to be a direct cause of this optic nervedamage.

Early work in the field of glaucoma research has indicated that elevatedIOP leads to interference in axonal transport at the level of the laminacribosa (a perforated, collagenous connective tissue) that is followedby the death of retinal ganglion cells. Quigley and Anderson (1976)Invest. Ophthalmol. Vis. Sci., 15, 606-16; Minckler, Bunt, and Klock,(1978) Invest. Ophthalmol. Vis. Sci., 17, 33-50; Anderson andHendrickson, (1974) Invest. Ophthalmol. Vis. Sci., 13, 771-83; Quigleyet al., (1980) Invest. Ophthalmol. Vis. Sci., 19, 505-17. Studies ofanimal models of glaucoma and post-mortem human tissues indicate thatthe death of retinal ganglion cells in glaucoma occurs by apoptosis.Garcia-Valenzuela et. al., (1995) Exp. Eye Res., 61, 33-44; Quigley etal., (1995) Invest. Ophthalmol. Vis. Sci., 36, 774-786; Monard, (1998)In: Haefliger Flammer J (eds) Nitric Oxide and Endothelin in thePathogenesis of Glaucoma, New York, N.Y., Lippincott-Raven, 213-220. Theinterruption of axonal transport as a result of increased IOP maycontribute to retinal ganglion cell death by deprivation of trophicfactors. Quigley, (1995) Aust N Z J Ophthalmol, 23(2), 85-91. Opticnerve head astrocytes in glaucomatous eyes have also been found toproduce increased levels of some neurotoxic substances. For example,increased production of tumor necrosis factor-α (TNF-α) (Yan et al.,(2000) Arch. Ophthalmol., 118, 666-673), and nitric oxide synthase(Neufeld et al., (1997) Arch. Ophthalmol., 115, 497-503), the enzymewhich gives rise to nitric oxide, has been found in the optic nerve headof glaucomatous eyes. Furthermore, increased expression of the inducibleform of nitric oxide synthase (iNOS) and TNF-α by activated retinalglial cells have been observed in rat models of hereditary retinaldiseases. Cotinet et al., (1997) Glia, 20, 59-69; de Kozak et al.,(1997) Ocul. Immunol. Inflamm., 5, 85-94; Goureau et al., (1999) J.Neurochem, 72, 2506-2515. In the glaucomatous optic nerve head,excessive nitric oxide has been linked to the degeneration of axons ofretinal ganglion cells. Arthur and Neufeld, (1999) Surv Ophthalmol, 43(Suppl 1), S129-S135. Finally, increased production of TNF-α by retinalglial cells in response to simulated ischemia or elevated hydrostaticpressure has been shown to induce apoptosis in cocultured retinalganglion cells. Tezel and Wax, (2000) J. Neurosci., 20(23), 8693-8700.

Protecting retinal ganglion cells from degeneration by apoptosis isunder study as a potential new treatment for blindness due to glaucoma.Antisense oligonucleotides have been used by several groups to targetkey proteins in the apoptotic process in order to protect retinalganglion cells from apoptotic cell death. Antisense oligonucleotides areshort, synthetic strands of DNA (or DNA analogs) that are antisense (orcomplimentary) to a specific DNA or RNA target. Antisenseoligonucleotides are designed to block expression of the protein encodedby the DNA or RNA target by binding to the target and halting expressionat the level of transcription, translation, or splicing. One of thehurdles to using antisense oligonucleotides as a drug is the rapiddegradation of oligonucleotides in blood and in cells by nucleases. Thisproblem has been addressed by using modified backbones that resistdegradation (Blake et al., (1985) Biochemistry, 24, 6139-6145) such asreplacement of the phosphodiester bonds in the oligonucleotides withphosphorothioate linkages to retard nuclease degradation. Matzura andEckstein, (1968) Eur. J. Biochem., 3, 448-452.

Antisense oligonucleotides have been used successfully in animal modelsof eye disease. In a model of transient global retinal ischemia,expression of caspase 2 was increased during ischemia, primarily in theinner nuclear and ganglion cell layers of the retina. Suppression ofcaspase using an antisense oligonucleotide led to significanthistopathologic and functional improvement as determined byelectroretinogram. Singh et al., (2001) J. Neurochem., 77(2), 466-75.Another study demonstrated that, upon transection of the optic nerve,retinal ganglion cells upregulate the pro-apoptotic protein Bax andundergo apoptosis. Repeated injections of a Bax antisenseoligonucleotide into the temporal superior retina of rats inhibited thelocal expression of Bax and increased the number of surviving retinalganglion cells following transaction of the optic nerve. Isenmann etal., (1999) Cell Death Differ., 6(7). 673-82.

Delivery of antisense oligonucleotides to retinal ganglion cells hasbeen improved by encapsulating the oligonucleotides in liposomes, whichwere then coated with the envelope of inactivated hemagglutinating virusof Japan (HVJ; Sendai virus) by fusion (HVJ liposomes). Intravitrealinjection into mice of FITC-labeled antisense oligonucleotidesencapsulated in HVJ liposomes resulted in high fluorescence within 44%of the cells in the ganglion layer which lasted three days whilefluorescence with naked FITC-labeled antisense oligonucleotidedisappeared after one day. Hangai et al., (1998) Arch Ophthalmol,116(7), 976.

A method of preventing or modulating apoptosis of the present inventionis directed to modulating apoptosis in the cells of the eye, such as butnot limited to, astrocytes, retinal ganglion, retinal glial cells andlamina cribosa. Death of retinal ganglion cells in glaucoma occurs byapoptosis. Thus, providing a method of inhibiting apoptosis in retinalganglion cells or by protecting retinal ganglion cells from degenerationby apoptosis provides a novel treatment for prevention of blindness dueto glaucoma.

The present invention provides a method for preventing retinal ganglioncell death in a glaucomatous eye, by suppressing expression ofapoptosis-specific eIF-5A1. Inhibiting the expression ofapoptosis-specific eIF-5A1 reduces apoptosis. Apoptosis-specific eIF-5A1is a powerful gene that appears to regulate the entire apoptic process.Thus, controlling apoptosis in the optic nerve head indicates thatblocking expression of apoptosis-specific eIF-5A1 provides a treatmentfor glaucoma.

Suppression of expression of apoptosis-specific eIF-5A1 is accomplishedby administering antisense oligonucleotides targeted against humanapoptosis-specific 5A1 to cells of the eye such as, but not limited tolamina crobosa, astrocytes, retinal ganglion, or retinal glial cells.Antisense oligonucleotides are as defined above, i.e. have a nucleotidesequence encoding at least a portion of an apoptosis-specific eIF-5A1polypeptide. Antisense oligonucleotides targeted against humanapoptosis-specific eIF-5A1 have a nucleotide sequence encoding at leasta portion of human apoptosis-specific eIF-5A1 polypeptide. Preferredantisense oligonucleotides comprise SEQ ID NO:26 or 27 oroligonucleotides that bind to a sequence complementary to SEQ ID NO:26or 27 under high stringency conditions.

Another embodiment of the invention provides a method of suppressingexpression of apoptosis-specific eIF-5A1 in lamina cribosa cells,astrocyte cells, retinal ganglion cells or retinal glial cells.Antisense oligonucleotides, such as but not limited to, SEQ ID NO:26 and27, targeted against human apoptosis-specific eIF-5A1 are administeredto lamina cribosa cells, astrocyte cells, retinal ganglion cells orretinal glial cells. The cells may be human.

In addition to having a role in apoptosis, eIF-5A may also have a rolein the immune response. The present inventors have discovered thatapoptosis factor 5A levels correlate with elevated levels of twocytokines (Interleukin 1-beta “IL-1β” and interleukin 18 “IL-18”) inischemic heart tissue, thus further proving that apoptosis factor 5A isinvolved in cell death as it is present in ischemic heart tissue.Further, this apoptosis factor 5A/interleukin correlation has not beenseen in non-ischemic heart tissue. See FIGS. 50A-F and 51. Using PCRmeasurements, levels of apoptosis factor 5A, proliferating eIF-5a(eIF-5A2—the other known isoform), IL-1β, and IL-18 were measured andcompared in various ischemic heart tissue (from coronary bypass graftand valve (mitral and atrial valve) replacement patients).

The correlation of apoptosis eIF-5a to these potent interleukins furthersuggests that the inflammation and apoptosis pathways in ischemia may becontrolled via controlling levels of apoptosis factor 5A. Furtherevidence that apoptosis factor 5A is involved in the immune response issuggested by the fact that human peripheral blood mononuclear cells(PBMCs) normally express very low levels of eIF-5A, but upon stimulationwith T-lymphocyte-specific stimuli expression of eIF-5A increasesdramatically (Bevec et al., 1994). This suggests a role for apoptosisfactor 5A in T-cell proliferation and/or activation. Since activated Tcells are capable of producing a wide variety of cytokines, it is alsopossible that apoptosis factor 5A may be required as a nucleocytoplasmicshuttle for cytokine mRNAs.

Another study looked at eIf-5A mRNA and cell surface marker expressionin human peripheral blood mononuclear cells (PBMCs) and blood cell linestreated with various maturation stimulating agents (Bevec et al., Proc.Natl. Acad. Sci. USA, 91:10829-10833. (1994)). eIF-5A mRNA expressionwas induced in the PBMCs by numerous stimuli that also induced T-cellactivation (Bevec et al., 1994). Higher levels of PBMC eIF 5A mRNAexpression were observed in HIV-1 patients than in healthy donors. Theauthors of this study interpreted their results by suggesting that theeIF-5A mRNA was induced so that it could act as a nucleocytoplasmicshuttle for the important mRNAs necessary for T-cell activation and alsofor HIV-1 replication (Bevec et al., 1994). eIF-5A has been demonstratedto be a cellular binding factor for the HIV Rev protein and required forHIV replication (Ruhl et al. 1993).

Recent studies have suggested an important role for eIF-5A in thedifferentiation and activation of cells. When immature dendritic cellswere induced to differentiate and mature, an induction of eIF-5A mRNAlevels coincided with an elevation of CD83 protein expression (Kruse etal., J. Exp. Med. 191(9): 1581-1589 (2000)). Dendritic cells areantigen-presenting cells that sensitize helper and killer T cells toinduce T cell-mediated immunity (Steinman, 1991). Immature dendriticcells lack the ability to stimulate T cells and require appropriatestimuli (i.e. inflammatory cytokines and/or microbial products) tomature into cells capable of activating T cells. The synthesis andsurface expression of CD83 on mature dendritic cells is importantlyinvolved in sensitizing helper and killer T cells and in inducing Tcell-mediated immunity. When the immature dendritic cells werepre-treated with an inhibitor (GC7) of hypusination and thus aninhibitor of eIF-5A activation, the surface expression of CD83 wasprevented (Kruse et al., 2000). The authors of this study interpretedtheir results that the eIF-5A was essential for the nucleocytoplasmictranslocation of the CD83 mRNA and that by blocking hypusination andthus eIF-5A, CD83 expression and dendritic cell maturation were blocked(Kruse et al., 2000).

In both of these studies (Bevec et al., 1994; Kruse et al., 2000)implicating a role for eIF-5A in the immune system, the authors did notspecify or identify which isoform of eIF-5A they were examining, nor didthey have a reason to. As briefly discussed above, humans are known tohave two isoforms of eIF-5A, eIF-5A1 (apoptosis factor 5A1) and eIF-5A2,both encoded on separate chromosomes. Prior to the present inventors'discoveries it was believed that both of these isoforms werefunctionally redundant. The oligonucleotide described by Bevec et al.that was used to detect eIF-5A mRNA in stimulated PBMCs had 100%homology to human elEF-5A1 and the study pre-dates the cloning ofeIF-5A2. Similarly, the primers described by Kruse et al. that were usedto detect eIF-5A by reverse transcription polymerase chain reactionduring dendritic cell maturation had 100% homology to human eIF-5A1.

The present invention relates to controlling the expression of eIF-5A1to control the rate of dendritic cell maturation and PBMC activation,which in turn may control the rate of T cell-mediated immunity. Thepresent inventors studied the role of eIF-5A1 in the differentiation ofmonocytes into adherent macrophages using the U-937 cell line, as U-937is known to express eIF-5A mRNA (Bevec et al., 1994). U-937 is a humanmonocyte cell line that grows in suspension and will become adherent anddifferentiate into macrophages upon stimulation with PMA. When PMA isremoved by changing the media, the cells become quiescent and are thencapable of producing cytokines (Barrios-Rodiles et al., J. Immunol.163:963-969 (1999)). In response to lipopolysaccharide (LPS), a factorfound on the outer membrane of many bacteria known to induce a generalinflammatory response, the macrophages produce both TNF-α and IL-113(Barrios-Rodiles et al., 1999). See FIG. 78 showing a chart of stem celldifferentiation and the resultant production of cytokines. The U-937cells also produce IL-6 and IL-10 following LPS-stimulation (Izeboud etal., J. Receptor & Signal Transduction Research, 19(1-4):191-202.(1999)).

Using U-937 cells, it was shown that eIF-5A1 is upregulated duringmonocyte differentiation and TNF-α secretion. See FIG. 77. Accordingly,one aspect of the invention provides for a method of inhibiting ordelaying maturation of macrophages to inhibit or reduce the productionof cytokines. This method involves providing an agent that is capable ofreducing the expression of either DHS or eIF-5A1. By reducing oreliminating expression of DHS, eIF-5A1 activation will be reduced oreliminated. Since, eIF-5A1 is upregulated during monocytedifferentiation and TNF-α secretion, it is believed that it is necessaryfor these events to occur. Thus, by reducing or eliminating activationof eIF-5A1 or by directly reducing or eliminating eIF-5A1 expression,monocyte differentiation and TNF-α secretion can be reduced oreliminated. Any agent capable of reducing the expression of DHS oreIF-5A1 may be used and includes, but is not limited to antisenseoligonucleotides or siRNAs as described herein.

The present inventors have studied the ability of eIF-5A1 to promotetranslation of cytokines by acting as a nucleocytoplasmic shuttle forcytokine mRNAs in vitro using a cell line known to predictably producecytokine(s) in response to a specific stimulus. Some recent studies havefound that human liver cell lines can respond to cytokine stimulation byinducing production of other cytokines. HepG2 is a well characterizedhuman hepatocellular carcinoma cell line found to be sensitive tocytokines. In response to IL-1β, HepG2 cells rapidly produce TNF-α mRNAand protein in a dose-dependent manner (Frede et al., 1996; Rowell etal., 1997; Wordemann et al., 1998). Thus, HepG2 cells were used as amodel system to study the regulation of TNF-α production. The presentinventors have shown that inhibition of eIF-5A1 expression in HepG2cells caused the cells to produce less TNF-α after having beentransfected with antisense nucleotides directed toward apoptosis factor5A.

Thus one embodiment of the present invention provides a method forreducing levels of a cytokine in a cell. The method involvesadministering an agent to the cell capable of reducing expression ofapoptosis factor 5A1. Reducing expression of apoptosis factor 5A1 causesa reduction in the expression of the cytokine, and thus leads to adecreased amount of the cytokine produced by cell. The cytokine is apreferably a pro-inflammatory cytokine, including, but not limited toIL-1, IL-18, IL-6 and TNF-α.

An agent capable of reducing expression of apoptosis factor 5A may be anantisense nucleotide having a sequence complementary to apoptosis factor5A. Preferably the antisense nucleotide has a sequence selected from thegroup consisting of SEQ ID NO: 63-65 or is an antisense nucleotide thathybridizes under highly stringent conditions to a sequence selected fromthe group consisting of SEQ ID NO: 63-65.

An agent may also comprise a siRNA having a sequence complementary toapoptosis factor 5A. Preferably the siRNA has a sequence selected fromthe group consisting of SEQ ID NO: 30, 31, 32, and 33, or is a siRNAthat hybridizes under highly stringent conditions to a sequence selectedfrom the group consisting of SEQ ID NO: 30, 31, 32, and 33. FIGS. 65-67show that cells transfected with apoptosis factor 5A siRNAs have a lowerpercentage of cells undergoing apoptosis after exposure to camptothecinand TNF-α. An agent may also comprise antisense DHS nucleotides.

The present invention is also directed to a polynucleotide having asequence selected from the group consisting of SEQ ID NO: 63-65 or is anantisense nucleotide that hybridizes under highly stringent conditionsto a sequence selected from the group consisting of SEQ ID NO: 63-65.

The present invention is also directed to a siRNA having a sequenceselected from the group consisting of SEQ ID NO: 30, 31, 32, and 33 oris a siRNA that hybridizes under highly stringent conditions to asequence selected from the group consisting of SEQ ID NO: 30, 31, 32,and 33.

The present invention is also directed to a method for reducing theexpression of p53. This method involves administering an agent capableof reducing expression of apoptosis factor 5A, such as the antisensepolynucleotides or the siRNAs described above. Reducing expression ofapoptosis factor 5A1 reduces expression of p53 as shown in FIG. 52 andexample 11.

The present invention is also directed to a method for increasing theexpression of Bcl-2. This method entails administering an agent capableof reducing expression of apoptosis factor 5A. Preferred agents includethe antisense oligonucleotides and siRNAs described above. Reducing ofexpression of apoptosis factor 5A1 increases expression of Bcl-2 asshown in FIG. 63 and example 13. FIG. 63 shows that cells transfectedwith apoptosis factor 5a siRNA produced less apoptosis factor 5A1protein and in addition, produced more Bcl-2 protein. Decrease inapoptosis factor 5A1 expression correlates with an increase in BCL-2expression.

The present invention also provides a method for reducing levels ofTNF-alpha in a patient in need thereof comprising administering to saidpatient either the antisense polynucleotide or siRNAs described above.As demonstrated in FIG. 69 and example 14, cells transfected withantisense factor 5A oligonucleotides of the present invention producedless TNF-α after induction with IL-1 than cells not transfected withsuch antisense oligonucleotides.

The present invention provides for a method of reducing levels ofTNF-alpha in human epithelial cells. As demonstrated in FIGS. 74A and Band FIG. 75 and example 15, reducing or inhibiting the expression ofeIF-5A1 causes a decrease, if not complete inhibition of the productionof TNF-alpha in a human epithelial cell line. siRNAs against eIF-5A1were used to inhibit expression of eIF-5A1. This inhibition ofexpression not only reduced or inhibited the production of TNF-alpha,but it also protected the cells from cytokine-induced apoptosis. Byreducing expression of eIF-5A1, the production of TNF-α is reduced. Thisdual effect provides a method of treating patients suffering frominflammatory bowel disorders such as Crohn's disease and ulcerativecolitis, which are associated with an increased inflammation caused byTNF-α.

Thus, the present invention provides a method of treating pathologicalconditions characterized by an increased IL-1, TNF-alpha, IL-6 or IL-18level comprising administering to a mammal having said pathologicalcondition, an agent to reduce expression of apoptosis Factor 5A.

Known pathological conditions characterized by an increase in IL-1,TNF-alpha, or Il-6 levels include, but are not limited to,arthritis-rheumatoid and osteo arthritis, asthma, allergies, arterialinflammation, Crohn's disease, inflammatory bowel disease (ibd),ulcerative colitis, coronary heart disease, cystic fibrosis, diabetes,lupus, multiple sclerosis, graves disease, periodontitis, glaucoma &macular degeneration, ocular surface diseases including keratoconus,organ ischemia-heart, kidney, repurfusion injury, sepsis, multiplemyeloma, organ transplant rejection, psoriasis and eczema.

As used herein, the term “substantial sequence identity” or “substantialhomology” is used to indicate that a sequence exhibits substantialstructural or functional equivalence with another sequence. Anystructural or functional differences between sequences havingsubstantial sequence identity or substantial homology will be deminimus; that is, they will not affect the ability of the sequence tofunction as indicated in the desired application. Differences may be dueto inherent variations in codon usage among different species, forexample. Structural differences are considered de minimus if there is asignificant amount of sequence overlap or similarity between two or moredifferent sequences or if the different sequences exhibit similarphysical characteristics even if the sequences differ in length orstructure. Such characteristics include, for example, the ability tohybridize under defined conditions, or in the case of proteins,immunological crossreactivity, similar enzymatic activity, etc. Theskilled practitioner can readily determine each of these characteristicsby art known methods.

Additionally, two nucleotide sequences are “substantially complementary”if the sequences have at least about 70 percent or greater, morepreferably 80 percent or greater, even more preferably about 90 percentor greater, and most preferably about 95 percent or greater sequencesimilarity between them. Two amino acid sequences are substantiallyhomologous if they have at least 50%, preferably at least 70%, morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 95% similarity between the active, or functionallyrelevant, portions of the polypeptides.

To determine the percent identity of two sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, at least 30%, 40%,50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequenceis aligned for comparison purposes. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram. BLAST protein searches can be performed with the)(BLAST programto obtain amino acid sequences homologous to the proteins of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and gapped BLASTprograms, the default parameters of the respective programs (e.g.,(BLAST and NBLAST) can be used.

The term “functional derivative” of a nucleic acid is used herein tomean a homolog or analog of the gene or nucleotide sequence. Afunctional derivative may retain at least a portion of the function ofthe given gene, which permits its utility in accordance with theinvention. “Functional derivatives” of the apoptosis factor 5Apolypeptide as described herein are fragments, variants, analogs, orchemical derivatives of apoptosis factor 5A that retain at least aportion of the apoptosis factor 5A activity or immunological crossreactivity with an antibody specific for apoptosis factor 5A. A fragmentof the apoptosis factor 5A polypeptide refers to any subset of themolecule.

Functional variants can also contain substitutions of similar aminoacids that result in no change or an insignificant change in function.Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al. (1989) Science 244:1081-1085). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et al.(1992) Science 255:306-312).

A “variant” refers to a molecule substantially similar to either theentire gene or a fragment thereof, such as a nucleotide substitutionvariant having one or more substituted nucleotides, but which maintainsthe ability to hybridize with the particular gene or to encode mRNAtranscript which hybridizes with the native DNA. A “homolog” refers to afragment or variant sequence from a different animal genus or species.An “analog” refers to a non-natural molecule substantially similar to orfunctioning in relation to the entire molecule, a variant or a fragmentthereof.

Variant peptides include naturally occurring variants as well as thosemanufactured by methods well known in the art. Such variants can readilybe identified/made using molecular techniques and the sequenceinformation disclosed herein. Further, such variants can readily bedistinguished from other proteins based on sequence and/or structuralhomology to the eIF-5A or DHS proteins of the present invention. Thedegree of homology/identity present will be based primarily on whetherthe protein is a functional variant or non-functional variant, theamount of divergence present in the paralog family and the evolutionarydistance between the orthologs.

Non-naturally occurring variants of the eIF-5A or DHS proteins of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the proteins. For example,one class of substitutions are conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in a proteinby another amino acid of like characteristics. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr; exchange of the acidic residues Asp andGlu; substitution between the amide residues Asn and Gln; exchange ofthe basic residues Lys and Arg; and replacements among the aromaticresidues Phe and Tyr. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247:1306-1310 (1990).

The term “hybridization” as used herein is generally used to meanhybridization of nucleic acids at appropriate conditions of stringencyas would be readily evident to those skilled in the art depending uponthe nature of the probe sequence and target sequences. Conditions ofhybridization and washing are well known in the art, and the adjustmentof conditions depending upon the desired stringency by varyingincubation time, temperature and/or ionic strength of the solution arereadily accomplished. See, e.g. Sambrook, J. et al., Molecular Cloning:A Laboratory Manual, 2^(nd) edition, Cold Spring Harbour Press, ColdSpring Harbor, N.Y., 1989.

The choice of conditions is dictated by the length of the sequencesbeing hybridized, in particular, the length of the probe sequence, therelative G-C content of the nucleic acids and the amount of mismatchesto be permitted. Low stringency conditions are preferred when partialhybridization between strands that have lesser degrees ofcomplementarity is desired. When perfect or near perfect complementarityis desired, high stringency conditions are preferred. For typical highstringency conditions, the hybridization solution contains 6×S.S.C.,0.01 M EDTA, 1×Denhardt's solution and 0.5% SDS. Hybridization iscarried out at about 68° C. for about 3 to 4 hours for fragments ofcloned DNA and for about 12 to 16 hours for total eucaryotic DNA. Forlower stringencies, the temperature of hybridization is reduced to about42° C. below the melting temperature (T_(m)) of the duplex. The T_(m) isknown to be a function of the G-C content and duplex length as well asthe ionic strength of the solution.

As used herein, the phrase “hybridizes to a corresponding portion” of aDNA or RNA molecule means that the molecule that hybridizes, e.g.,oligonucleotide, polynucleotide, or any nucleotide sequence (in sense orantisense orientation) recognizes and hybridizes to a sequence inanother nucleic acid molecule that is of approximately the same size andhas enough sequence similarity thereto to effect hybridization underappropriate conditions. For example, a 100 nucleotide long sensemolecule will recognize and hybridize to an approximately 100 nucleotideportion of a nucleotide sequence, so long as there is about 70% or moresequence similarity between the two sequences. It is to be understoodthat the size of the “corresponding portion” will allow for somemismatches in hybridization such that the “corresponding portion” may besmaller or larger than the molecule which hybridizes to it, for example20-30% larger or smaller, preferably no more than about 12-15% larger orsmaller.

In addition, functional variants of polypeptides can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Amino acids that are essential forfunction can be identified by methods known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunningham etal., Science 244:1081-1085 (1989)). The latter procedure introducessingle alanine mutations at every residue in the molecule. The resultingmutant molecules are then tested for biological activity or in assays.

For example, an analog of apoptosis factor 5A refers to a non-naturalprotein or peptidomimetic substantially similar to either the entireprotein or a fragment thereof. Chemical derivatives of apoptosis factor5A contain additional chemical moieties not normally a part of thepeptide or peptide fragment. Modifications can be introduced intopeptide or fragment thereof by reacting targeted amino acid residues ofthe peptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues.

It is understood that the nucleic acids and polypeptides of the presentinvention, where used in an animal for the purpose of prophylaxis ortreatment, will be administered in the form of a compositionadditionally comprising a pharmaceutically acceptable carrier. Suitablepharmaceutically acceptable carriers include, for example, one or moreof water, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. Pharmaceuticallyacceptable carriers can further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the bindingproteins. The compositions of the injection can, as is well known in theart, be formulated so as to provide quick, sustained or delayed releaseof the active ingredient after administration to the mammal.

The compositions of this invention can be in a variety of forms. Theseinclude, for example, solid, semi-solid and liquid dosage forms, such astablets, pills, powders, liquid solutions, dispersions or suspensions,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application.

Such compositions can be prepared in a manner well known in thepharmaceutical art. In making the composition the active ingredient willusually be mixed with a carrier, or diluted by a carrier, and/orenclosed within a carrier which can, for example, be in the form of acapsule, sachet, paper or other container. When the carrier serves as adiluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, excipient or medium for the active ingredient. Thus, thecomposition can be in the form of tablets, lozenges, sachets, cachets,elixirs, suspensions, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, injectionsolutions, suspensions, sterile packaged powders and as a topical patch.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration. The Examples are set forth to aidin understanding the invention but are not intended to, and should notbe construed to, limit its scope in any way. The examples do not includedetailed descriptions of conventional methods. Such methods are wellknown to those of ordinary skill in the art and are described innumerous publications. Detailed descriptions of conventional methods,such as those employed in the construction of vectors and plasmids, theinsertion of nucleic acids encoding polypeptides into such vectors andplasmids, the introduction of plasmids into host cells, and theexpression and determination thereof of genes and gene products can beobtained from numerous publication, including Sambrook, J. et al.,(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press. All references mentioned herein areincorporated in their entirety.

EXAMPLES Example 1 Visualization of Apoptosis in Rat Corpus Luteum byDNA Laddering

The degree of apoptosis was determined by DNA laddering. Genomic DNA wasisolated from dispersed corpus luteal cells or from excised corpusluteum tissue using the QIAamp DNA Blood Kit (Qiagen) according to themanufacturer's instructions. Corpus luteum tissue was excised before theinduction of apoptosis by treatment with PGF-2α, 1 and 24 hours afterinduction of apoptosis. The isolated DNA was end-labeled by incubating500 ng of DNA with 0.2 μCi [α-³²P]dCTP, 1 mM Tris, 0.5 mM EDTA, 3 unitsof Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTP at roomtemperature for 30 minutes. Unincorporated nucleotides were removed bypassing the sample through a 1 ml Sephadex G-50 column according toSambrook et al. The samples were then resolved by Tris-acetate-EDTA(1.8%) gel electrophoresis. The gel was dried for 30 minutes at roomtemperature under vacuum and exposed to x-ray film at −80° C. for 24hours.

In one experiment, the degree of apoptosis in superovulated rat corpuslutea was examined either 0, 1, or 24 hours after injection with PGF-2α.In the 0 hour control, the ovaries were removed without PGF-2αinjection. Laddering of low molecular weight DNA fragments reflectingnuclease activity associated with apoptosis is not evident in controlcorpus luteum tissue excised before treatment with PGF-2α, but isdiscernible within 1 hour after induction of apoptosis and is pronouncedby 24 hours after induction of apoptosis, which is shown in FIG. 16. Inthis figure, the top panel is an autoradiograph of the Northern blotprobed with the ³²P-dCTP-labeled 3′-untranslated region of rat corpusluteum apoptosis-specific DHS cDNA. The lower panel is the ethidiumbromide stained gel of total RNA. Each lane contains 10 μg RNA. The dataindicates that there is down-regulation of eIF-5A transcript followingserum withdrawal.

In another experiment, the corresponding control animals were treatedwith saline instead of PGF-2α. Fifteen minutes after treatment withsaline or PGF-2α, corpora lutea were removed from the animals. GenomicDNA was isolated from the corpora lutea at 3 hours and 6 hours afterremoval of the tissue from the animals. DNA laddering and increased endlabeling of genomic DNA are evident 6 hours after removal of the tissuefrom the PGF-2α-treated animals, but not at 3 hours after removal of thetissue. See FIG. 17. DNA laddering reflecting apoptosis is also evidentwhen corpora lutea are excised 15 minutes after treatment with PGF-2αand maintained for 6 hours under in vitro conditions in EBSS (Gibco).Nuclease activity associated with apoptosis is also evident from moreextensive end labeling of genomic DNA.

In another experiment, superovulation was induced by subcutaneousinjection with 500 μg of PGF-2α. Control rats were treated with anequivalent volume of saline solution. Fifteen to thirty minutes later,the ovaries were removed and minced with collagenase. The dispersedcells from rats treated with PGF-2α were incubated in 10 mm glutamine+10mm spermidine for 1 hour and for a further 5 hours in 10 mm glutaminewithout spermidine (lane 2) or in 10 mm glutamine+10 mm spermidine for 1hour and for a further 5 hours in 10 mm glutamine+1 mm spermidine (lane3). Control cells from rats treated with saline were dispersed withcollagenase and incubated for 1 hour and a further 5 hours in glutamineonly (lane 1). Five hundred nanograms of DNA from each sample waslabeled with [α-³²P]-dCTP using Klenow enzyme, separated on a 1.8%agarose gel, and exposed to film for 24 hours. Results are shown in FIG.18.

In yet another experiment, superovulated rats were injectedsubcutaneously with 1 mg/100 g body weight of spermidine, delivered inthree equal doses of 0.333 mg/100 g body weight, 24, 12, and 2 hoursprior to a subcutaneous injection with 500 μg PGF-2α. Control rats weredivided into three sets: no injections, three injections of spermidinebut no PGF-2α; and three injections with an equivalent volume of salineprior to PGF-2α treatment. Ovaries were removed front the rats either 1hour and 35 minutes or 3 hours and 45 minutes after prostaglandintreatment and used for the isolation of DNA. Five hundred nanograms ofDNA from each sample was labeled with [α-³²P]-dCTP using Klenow enzyme,separated on a 1.8% agarose gel, and exposed to film for 24 hours: lane1, no injections (animals were sacrificed at the same time as for lanes3-5); lane 2, three injections with spermidine (animals were sacrificedat the same time as for lanes 3-5); lane 3, three injections with salinefollowed by injection with PGF-2α (animals were sacrificed 1 h and 35min after treatment with PGF-2α); lane 4, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 1h and 35 min after treatment with PGF-2α); lane 5, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 1h and 35 min after treatment with PGF-2α); lane 6, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 3h and 45 min after treatment with PGF-2α); lane 7, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 3h and 45 min after treatment with PGF-2α). Results are shown in FIG. 19.

RNA Isolation

Total RNA was isolated from corpus luteum tissue removed from rats atvarious times after PGF-2α induction of apoptosis. Briefly, the tissue(5 g) was ground in liquid nitrogen. The ground powder was mixed with 30ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH8.5, 0.8% β-mercaptoethanol). The mixture was filtered through fourlayers of Miracloth and centrifuged at 10,000 g at 4° C. for 30 minutes.The supernatant was then subjected to cesium chloride density gradientcentrifugation at 11,200 g for 20 hours. The pelleted RNA was rinsedwith 75% ethanol, resuspended in 600 ml DEPC-treated water and the RNAprecipitated at −70° C. with 1.5 ml 95% ethanol and 60 ml of 3M NaOAc.

Genomic DNA Isolation and Laddering

Genomic DNA was isolated from extracted corpus luteum tissue ordispersed corpus luteal cells using the QIAamp DNA Blood Kit (Qiagen)according to the manufacturer's instructions. The DNA was end-labeled byincubating 500 ng of DNA with 0.2 μCi [α-³²P]dCTP, 1 mM Tris, 0.5 mMEDTA, 3 units of Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTP,at room temperature for 30 minutes. Unincorporated nucleotides wereremoved by passing the sample through a 1-ml Sephadex G-50 columnaccording to the method described by Maniatis et al. The samples werethen resolved by Tris-acetate-EDTA (2%) gel electrophoresis. The gel wasdried for 30 minutes at room temperature under vacuum and exposed tox-ray film at −80° C. for 24 hours.

Plasmid DNA Isolation, DNA Sequencing

The alkaline lysis method described by Sambrook et al., supra, was usedto isolate plasmid DNA. The full-length positive cDNA clone wassequenced using the dideoxy sequencing method. Sanger et al., Proc.Natl. Acad. Sci. USA, 74:5463-5467. The open reading frame was compiledand analyzed using BLAST search (GenBank, Bethesda, Md.) and sequencealignment was achieved using a BCM Search Launcher: Multiple SequenceAlignments Pattern-Induced Multiple Alignment Method (see F. Corpet,Nuc. Acids Res., 16:10881-10890, (1987). Sequences and sequencealignments are shown in FIGS. 5-11.

Northern Blot Hybridization of Rat Corpus Luteum RNA

Twenty milligrams of total RNA isolated from rat corpus luteum atvarious stages of apoptosis were separated on 1% denatured formaldehydeagarose gels and immobilized on nylon membranes. The full-length ratapoptosis-specific eIF-5A cDNA (SEQ ID NO:1) labeled with ³²P-dCTP usinga random primer kit (Boehringer) was used to probe the membranes 7×10⁷.Alternatively, full length rat apoptosis-specific DHS cDNA (SEQ ID NO:6)labeled with ³²P-dCTP using a random primer kit (Boehringer) was used toprobe the membranes (7×10⁷ cpm). The membranes were washed once with1×SSC, 0.1% SDS at room temperature and three times with 0.2×SSC, 0.1%SDS at 65° C. The membranes were dried and exposed to X-ray filmovernight at −70° C.

As can be seen, eIF-5A and DHS are both upregulated in apoptosing corpusluteum tissue. Expression of apoptosis-specific eIF-5A is significantlyenhanced after induction of apoptosis by treatment with PGF-2α—low attime zero, increased substantially within 1 hour of treatment, increasedstill more within 8 hours of treatment and increased slightly within 24hours of treatment (FIG. 14). Expression of DHS was low at time zero,increased substantially within 1 hour of treatment, increased still morewithin 8 hours of treatment and increased again slightly within 24 hoursof treatment (FIG. 15).

Generation of an Apoptosing Rat Corpus Luteum RT-PCR Product UsingPrimers Based on Yeast, Fungal and Human eIF-5A Sequences

A partial-length apoptosis-specific eIF-5A sequence (SEQ ID NO:11)corresponding to the 3′ end of the gene was generated from apoptosingrat corpus luteum RNA template by RT-PCR using a pair of oligonucleotideprimers designed from yeast, fungal and human eIF-5A sequences. Theupstream primer used to isolate the 3′ end of the rat eIF-5A gene is a20 nucleotide degenerate primer: 5′ TCSAARACHGGNAAGCAYGG 3′ (SEQ IDNO:9), wherein S is selected from C and G; R is selected from A and G; His selected from A, T, and C; Y is selected from C and T; and N is anynucleic acid. The downstream primer used to isolate the 3′ end of therat eIF-5A gene contains 42 nucleotides: 5′ GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3′ (SEQ ID NO:10). A reverse transcriptasepolymerase chain reaction (RT-PCR) was carried out. Briefly, using 5 mgof the downstream primer, a first strand of cDNA was synthesized. Thefirst strand was then used as a template in a RT-PCR using both theupstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence of a 900 by fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using blunt end ligationand sequenced (SEQ ID NO:11). The cDNA sequence of the 3′ end is SEQ IDNO:11 and the amino acid sequence of the 3′ end is SEQ ID NO:12. SeeFIGS. 1-2.

A partial-length apoptosis-specific eIF-5A sequence (SEQ ID NO:15)corresponding to the 5′ end of the gene and overlapping with the 3′ endwas generated from apoptosing rat corpus luteum RNA template by RT-PCR.The 5′ primer is a 24-mer having the sequence, 5′CAGGTCTAGAGTTGGAATCGAAGC 3′ (SEQ ID NO:13), that was designed from humaneIF-5A sequences. The 3′ primer is a 30-mer having the sequence, 5′ATATCTCGAGCCTT GATTGCAACAGCTGCC 3′ (SEQ ID NO:14) that was designedaccording to the 3′ end RT-PCR fragment. A reversetranscriptase-polymerase chain reaction (RT-PCR) was carried out.Briefly, using 5 mg of the downstream primer, a first strand of cDNA wassynthesized. The first strand was then used as a template in a RT-PCRusing both the upstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 500 by fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using XbaI and XhoIcloning sites present in the upstream and downstream primers,respectively, and sequenced (SEQ ID NO:15). The cDNA sequence of the 5′end is SEQ ID NO:15, and the amino acid sequence of the 5′ end is SEQ IDNO:16. See FIG. 2.

The sequences of the 3′ and 5′ ends of the rat apoptosis-specific eIF-5A(SEQ ID NO:11 and SEQ ID NO:15, respectively) overlapped and gave riseto the full-length cDNA sequence (SEQ ID NO:1). This full-lengthsequence was aligned and compared with sequences in the GeneBank database. See FIGS. 1-2. The cDNA clone encodes a 154 amino acid polypeptide(SEQ ID NO:2) having a calculated molecular mass of 16.8 KDa. Thenucleotide sequence, SEQ ID NO:1, for the full length cDNA of the ratapoptosis-specific corpus luteum eIF-5A gene obtained by RT-PCR isdepicted in FIG. 3 and the corresponding derived amino acid sequence isSEQ ID NO:9. The derived full-length amino acid sequence of eIF-5A wasaligned with human and mouse eIF-5a sequences. See FIG. 7-9.

Generation of an Apoptosing Rat Corpus Luteum RT-PCR Product UsingPrimers Based on a Human DHS Sequence

A partial-length apoptosis-specific DHS sequence (SEQ ID NO:6)corresponding to the 3′ end of the gene was generated from apoptosingrat corpus luteum RNA template by RT-PCR using a pair of oligonucleotideprimers designed from a human DHS sequence. The 5′ primer is a 20-merhaving the sequence, 5′ GTCTGTGTATTATTGGGCCC 3′ (SEQ ID NO. 17); the 3′primer is a 42-mer having the sequence, 5′ GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3′ (SEQ ID NO:18). A reverse transcriptasepolymerase chain reaction (RT-PCR) was carried out. Briefly, using 5 mgof the downstream primer, a first strand of cDNA was synthesized. Thefirst strand was then used as a template in a RT-PCR using both theupstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence of a 606 by fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using blunt end ligationand sequenced (SEQ ID NO:6). The nucleotide sequence (SEQ ID NO:6) forthe partial length cDNA of the rat apoptosis-specific corpus luteum DHSgene obtained by RT-PCR is depicted in FIG. 4 and the correspondingderived amino acid sequence is SEQ ID NO. 7.

Isolation of Genomic DNA and Southern Analysis

Genomic DNA for southern blotting was isolated from excised rat ovaries.Approximately 100 mg of ovary tissue was divided into small pieces andplaced into a 15 ml tube. The tissue was washed twice with 1 ml of PBSby gently shaking the tissue suspension and then removing the PBS usinga pipette. The tissue was resuspended in 2.06 ml of DNA-buffer (0.2 MTris-HCl pH 8.0 and 0.1 mM EDTA) and 240 μl of 10% SDS and 100 μl ofproteinase K (Boehringer Manheim; 10 mg/ml) was added. The tissue wasplaced in a shaking water bath at 45° C. overnight. The following dayanother 100 μl of proteinase K (10 mg/ml) was added and the tissuesuspension was incubated in a water-bath at 45° C. for an additional 4hours. After the incubation the tissue suspension was extracted oncewith an equal volume of phenol:chloroform:iso-amyl alcohol (25:24:1) andonce with an equal volume of chloroform:iso-amyl alcohol (24:1).Following the extractions 1/10th volume of 3M sodium acetate (pH 5.2)and 2 volumes of ethanol were added. A glass pipette sealed and formedinto a hook using a Bunsen burner was used to pull the DNA threads outof solution and to transfer the DNA into a clean microcentrifuge tube.The DNA was washed once in 70% ethanol and air-dried for 10 minutes. TheDNA pellet was dissolved in 500 μl of 10 mM Tris-HCl (pH 8.0), 10 μl ofRNase A (10 mg/ml) was added, and the DNA was incubated for 1 hour at37° C. The DNA was extracted once with phenol:chloroform:iso-amylalcohol (25:24:1) and the DNA was precipitated by adding 1/10th volumeof 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The DNA waspelleted by centrifugation for 10 minutes at 13,000×g at 4° C. The DNApellet was washed once in 70% ethanol and dissolved in 200 μl 10 mMTris-HCl (pH 8.0) by rotating the DNA at 4° C. overnight.

For Southern blot analysis, genomic DNA isolated from rat ovaries wasdigested with various restriction enzymes that either do not cut in theendogenous gene or cut only once. To achieve this, 10 μg genomic DNA, 20μl 10× reaction buffer and 100 U restriction enzyme were reacted forfive to six hours in a total reaction volume of 200 μl. Digested DNA wasloaded onto a 0.7% agarose gel and subjected to electrophoresis for 6hours at 40 volts or overnight at 15 volts. After electrophoresis, thegel was depurinated for 10 minutes in 0.2 N HCl followed by two15-minute washes in denaturing solution (0.5 M NaOH, 1.5 M NaCl) and two15 minute washes in neutralizing buffer (1.5 M NaCl, 0.5 M Tris-HCl pH7.4). The DNA was transferred to a nylon membrane, and the membrane wasprehybridized in hybridization solution (40% formamide, 6×SSC,5×Denhart's, solution (1×Denhart's solution is 0.02% Ficoll, 0.02% PVP,and 0.02% BSA), 0.5% SDS, and 1.5 mg of denatured salmon sperm DNA). A700 by PCR fragment of the 3′ UTR of rat eIF-5A cDNA (650 by of 3′ UTRand 50 by of coding) was labeled with [a-32P]-dCTP by random priming andadded to the membrane at 1×10⁶ cpm/ml.

Similarly, a 606 by PCR fragment of the rat DHS cDNA (450 by coding and156 by 3′ UTR) was random prime labeled with [a-³²P]-dCTP and added at1×10⁶ cpm/ml to a second identical membrane. The blots were hybridizedovernight at 42° C. and then washed twice with 2×SSC and 0.1% SDS at 42°C. and twice with 1×SSC and 0.1% SDS at 42° C. The blots were thenexposed to film for 3-10 days.

Rat corpus genomic DNA was cut with restriction enzymes as indicated onFIG. 20 and probed with ³²P-dCTP-labeled eIF-5A cDNA. Hybridizationunder high stringency conditions revealed hybridization of thefull-length cDNA probe to several restriction fragments for eachrestriction enzyme digested DNA sample, indicating the presence ofseveral isoforms of eIF-5A. Of particular note, when rat genomic DNA wasdigested with EcoRV, which has a restriction site within the openreading frame of apoptosis-specific eIF-5A, two restriction fragments ofthe apoptosis-specific isoform of eIF-5A were detectable in the Southernblot. The two fragments are indicated with double arrows in FIG. 20. Therestriction fragment corresponding to the apoptosis-specific isoform ofeIF-5A is indicated by a single arrow in the lanes labeled EcoR1 andBamH1, restriction enzymes for which there are no cut sites within theopen reading frame. These results suggest that the apoptosis-specificeIF-5A is a single copy gene in rat. As shown in FIGS. 5 through 13, theeIF-5A gene is highly conserved across species, and so it would beexpected that there is a significant amount of conservation betweenisoforms within any species.

FIG. 21 shows a Southern blot of rat genomic DNA probed with³²P-dCTP-labeled partial-length rat corpus luteum apoptosis-specific DHScDNA. The genomic DNA was cut with EcoRV, a restriction enzyme that doesnot cut the partial-length cDNA used as a probe. Two restrictionfragments are evident indicating that there are two copies of the geneor that the gene contains an intron with an EcoRV site.

Example 2

The present example demonstrates modulation of apoptosis with apoptosisfactor 5A and DHS.

Culturing of COS-7 Cells and Isolation of RNA

COS-7, an African green monkey kidney fibroblast-like cell linetransformed with a mutant of SV40 that codes for wild-type T antigen,was used for all transfection-based experiments. COS-7 cells werecultured in Dulbecco's Modified Eagle's medium (DMEM) with 0.584 gramsper liter of L-glutamine, 4.5 g of glucose per liter, and 0.37% sodiumbicarbonate. The culture media was supplemented with 10% fetal bovineserum (FBS) and 100 units of penicillin/streptomycin. The cells weregrown at 37° C. in a humidified environment of 5% CO₂ and 95% air. Thecells were subcultured every 3 to 4 days by detaching the adherent cellswith a solution of 0.25% trypsin and 1 mM EDTA. The detached cells weredispensed at a split ratio of 1:10 in a new culture dish with freshmedia.

COS-7 cells to be used for isolation of RNA were grown in 150-mm tissueculture treated dishes (Corning). The cells were harvested by detachingthem with a solution of trypsin-EDTA. The detached cells were collectedin a centrifuge tube, and the cells were pelleted by centrifugation at3000 rpm for 5 minutes. The supernatant was removed, and the cell pelletwas flash-frozen in liquid nitrogen. RNA was isolated from the frozencells using the GenElute Mammalian Total RNA Miniprep kit (Sigma)according to the manufacturer's instructions.

Construction of Recombinant Plasmids and Transfection of COS-7 Cells

Recombinant plasmids carrying the full-length coding sequence of ratapoptosis eIF-5A in the sense orientation and the 3′ untranslated region(UTR) of rat apoptosis eIF-5A in the antisense orientation wereconstructed using the mammalian epitope tag expression vector, pHM6(Roche Molecular Biochemicals), which is illustrated in FIG. 21. Thevector contains the following: CMV promoter—human cytomegalovirusimmediate-early promoter/enhancer; HA—nonapeptide epitope tag frominfluenza hemagglutinin; BGH pA—Bovine growth hormone polyadenylationsignal; f1 ori—f1 origin; SV40 ori—SV40 early promoter and origin;Neomycin—Neomycin resistance (G418) gene; SV40 pA—SV40 polyadenylationsignal; Col E1—ColE1 origin; Ampicillin—Ampicillin resistance gene. Thefull-length coding sequence of rat apoptosis eIF-5A and the 3′ UTR ofrat apoptosis eIF-5A were amplified by PCR from the original rat eIF-5ART-PCR fragment in pBluescript (SEQ ID NO:1). To amplify the full-lengtheIF-5A the primers used were as follows: Forward 5′GCCAAGCTTAATGGCAGATGATTT GG 3′ (SEQ ID NO: 59) (Hind3) and Reverse 5′CTGAATTCCAGT TATTTTGCCATGG 3′ (SEQ ID NO: 60) (EcoR1). To amplify the 3′UTR rat eIF-5A the primers used were as follows: forward 5′AATGAATTCCGCCATGACAGAGGAGGC 3′ (SEQ ID NO: 61) (EcoR1) and reverse 5′GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3′ (SEQ ID NO: 62) (Hind3).

The full-length rat eIF-5A PCR product isolated after agarose gelelectrophoresis was 430 by in length while the 3′ UTR rat eIF-5A PCRproduct was 697 by in length. Both PCR products were subcloned into theHind 3 and EcoR1 sites of pHM6 to create pHM6-full-length eIF-5A andpHM6-antisense 3′UTReIF-5A. The full-length rat eIF-5A PCR product wassubcloned in frame with the nonapeptide epitope tag from influenzahemagglutinin (HA) present upstream of the multiple cloning site toallow for detection of the recombinant protein using ananti-[HA]-peroxidase antibody. Expression is driven by the humancytomegalovirus immediate-early promoter/enhancer to ensure high levelexpression in mammalian cell lines. The plasmid also features aneomycin-resistance (G418) gene, which allows for selection of stabletransfectants, and a SV40 early promoter and origin, which allowsepisomal replication in cells expressing SV40 large T antigen, such asCOS-7.

COS-7 cells to be used in transfection experiments were cultured ineither 24 well cell culture plates (Corning) for cells to be used forprotein extraction, or 4 chamber culture slides (Falcon) for cells to beused for staining. The cells were grown in DMEM media supplemented with10% FBS, but lacking penicillin/streptomycin, to 50 to 70% confluency.Transfection medium sufficient for one well of a 24-well plate orculture slide was prepared by diluting 0.32 μg of plasmid DNA in 42.5 μlof serum-free DMEM and incubating the mixture at room temperature for 15minutes. 1.6 μl of the transfection reagent, LipofectAMINE (Gibco, BRL),was diluted in 42.5 μl of serum-free DMEM and incubated for 5 minutes atroom temperature. After 5 minutes the LipofectAMINE mixture was added tothe DNA mixture and incubated together at room temperature for 30 to 60minutes. The cells to be transfected were washed once with serum-freeDMEM before overlaying the transfection medium and the cells were placedback in the growth chamber for 4 hours.

After the incubation, 0.17 ml of DMEM+20% FBS was added to the cells.The cells were then cultured for a further 40 hours before either beinginduced to undergo apoptosis prior to staining or harvested for Westernblot analysis. As a control, mock transfections were also performed inwhich the plasmid DNA was omitted from the transfection medium.

Protein Extraction and Western Blotting

Protein was isolated for Western blotting from transfected cells bywashing the cells twice in PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/LNa₂HPO₄, and 0.24 g/L KH₂PO₄) and then adding 150 μl of hot SDSgel-loading buffer (50 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2%SDS, 0.1% bromophenol blue, and 10% glycerol). The cell lysate wascollected in a microcentrifuge tube, heated at 95° C. for 10 minutes,and then centrifuged at 13,000×g for 10 minutes. The supernatant wastransferred to a fresh microcentrifuge tube and stored at −20° C. untilready for use.

For Western blotting, 2.5 or 5 μg of total protein was separated on a12% SDS-polyacrylamide gel. The separated proteins were transferred to apolyvinylidene difluoride membrane. The membrane was then incubated forone hour in blocking solution (5% skim milk powder, 0.02% sodium azidein PBS) and washed three times for 15 minutes in PBS-T (PBS+0.05%Tween-20). The membrane was stored overnight in PBS-T at 4° C. Afterbeing warmed to room temperature the next day, the membrane was blockedfor 30 seconds in 1 μg/ml polyvinyl alcohol. The membrane was rinsed 5times in deionized water and then blocked for 30 minutes in a solutionof 5% milk in PBS. The primary antibody was preincubated for 30 minutesin a solution of 5% milk in PBS prior to incubation with the membrane.

Several primary antibodies were used. An anti-[HA]-peroxidase antibody(Roche Molecular Biochemicals) was used at a dilution of 1:5000 todetect expression of the recombinant proteins. Since this antibody isconjugated to peroxidase, no secondary antibody was necessary, and theblot was washed and developed by chemiluminescence. The other primaryantibodies that were used are monoclonal antibodies from Oncogene thatrecognize p53 (Ab-6), Bcl-2 (Ab-1), and c-Myc (Ab-2). The monoclonalantibody to p53 was used at a dilution of 0.1 μg/ml, and the monoclonalantibodies to Bcl-2 and c-Myc were both used at a dilution of 0.83μg/ml. After incubation with primary antibody for 60 to 90 minutes, themembrane was washed 3 times for 15 minutes in PBS-T. Secondary antibodywas then diluted in 1% milk in PBS and incubated with the membrane for60 to 90 minutes. When p53 (Ab-6) was used as the primary antibody, thesecondary antibody used was a goat anti-mouse IgG conjugated to alkalinephosphatase (Rockland) at a dilution of 1:1000. When Bcl-2 (Ab-1) andc-Myc (Ab-2) were used as the primary antibody, a rabbit anti-mouse IgGconjugated to peroxidase (Sigma) was used at a dilution of 1:5000. Afterincubation with the secondary antibody, the membrane was washed 3 timesin PBS-T.

Two detection methods were used to develop the blots, a colorimetricmethod and a chemiluminescent method. The colorimetric method was usedonly when p53 (Ab-6) was used as the primary antibody in conjunctionwith the alkaline phosphatase-conjugated secondary antibody. Boundantibody was visualized by incubating the blot in the dark in a solutionof 0.33 mg/mL nitro blue tetrazolium, 0.165 mg/mL5-bromo-4-chloro-3-indolyl phosphate, 100 mM NaCl, 5 mM MgCl₂, and 100mM Tris-HO (pH 9.5). The color reaction was stopped by incubating theblot in 2 mM EDTA in PBS. A chemiluminescent detection method was usedfor all other primary antibodies, including anti-[HA]-peroxidase, Bcl-2(Ab-1), and c-Myc (Ab-2). The ECL Plus Western blotting detection kit(Amersham Pharmacia Biotech) was used to detect peroxidase-conjugatedbound antibodies. In brief, the membrane was lightly blotted dry andthen incubated in the dark with a 40:1 mix of reagent A and reagent Bfor 5 minutes. The membrane was blotted dry, placed between sheets ofacetate, and exposed to X-ray film for time periods varying from 10seconds to 10 minutes.

Induction of Apoptosis in COS 7 Cells

Two methods were used to induce apoptosis in transfected COS-7 cells,serum deprivation and treatment with Actinomycin D, streptomyces sp(Calbiochem). For both treatments, the medium was removed 40 hourspost-transfection. For serum starvation experiments, the media wasreplaced with serum- and antibiotic-free DMEM. Cells grown inantibiotic-free DMEM supplemented with 10% FBS were used as a control.For Actinomycin D induction of apoptosis, the media was replaced withantibiotic-free DMEM supplemented with 10% FBS and 1 μg/ml Actinomycin Ddissolved in methanol. Control cells were grown in antibiotic-free DMEMsupplemented with 10% FBS and an equivalent volume of methanol. For bothmethods, the percentage of apoptotic cells was determined 48 hours laterby staining with either Hoescht or Annexin V-Cy3. Induction of apoptosiswas also confirmed by Northern blot analyses, as shown in FIG. 22.

Hoescht Staining

The nuclear stain, Hoescht, was used to label the nuclei of transfectedCOS-7 cells in order to identify apoptotic cells based on morphologicalfeatures such as nuclear fragmentation and condensation. A fixative,consisting of a 3:1 mixture of absolute methanol and glacial aceticacid, was prepared immediately before use. An equal volume of fixativewas added to the media of COS-7 cells growing on a culture slide andincubated for 2 minutes. The media/fixative mixture was removed from thecells and discarded, and 1 ml of fixative was added to the cells. After5 minutes the fixative was discarded, and 1 ml of fresh fixative wasadded to the cells and incubated for 5 minutes. The fixative wasdiscarded, and the cells were air-dried for 4 minutes before adding 1 mlof Hoescht stain (0.5 μg/ml Hoescht 33258 in PBS). After a 10-minuteincubation in the dark, the staining solution was discarded and theslide was washed 3 times for 1 minute with deionized water. Afterwashing, 1 ml of McIlvaine's buffer (0.021 M citric acid, 0.058 MNa₂HPO₄.7H₂O; pH 5.6) was added to the cells, and they were incubated inthe dark for 20 minutes. The buffer was discarded, the cells wereair-dried for 5 minutes in the dark and the chambers separating thewells of the culture slide were removed. A few drops of Vectashieldmounting media for fluorescence (Vector Laboratories) was added to theslide and overlaid with a coverslip. The stained cells were viewed undera fluorescence microscope using a UV filter. Cells with brightly stainedor fragmented nuclei were scored as apoptotic.

Annexin V-Cy3 Staining

An Annexin V-Cy3 apoptosis detection kit (Sigma) was used tofluorescently label externalized phosphatidylserine on apoptotic cells.The kit was used according to the manufacturer's protocol with thefollowing modifications. In brief, transfected COS-7 cells growing onfour chamber culture slides were washed twice with PBS and three timeswith 1× Binding Buffer. 150 μl of staining solution (1 g/ml AnnCy3 in 1×Binding Buffer) was added, and the cells were incubated in the dark for10 minutes. The staining solution was then removed, and the cells werewashed 5 times with 1× Binding Buffer. The chamber walls were removedfrom the culture slide, and several drops of 1× Binding Buffer wereplaced on the cells and overlaid with a coverslip. The stained cellswere analyzed by fluorescence microscopy using a green filter tovisualize the red fluorescence of positively stained (apoptotic) cells.The total cell population was determined by counting the cell numberunder visible light.

Example 3

The present example demonstrates modulation of apoptosis with apoptosisfactor 5A and DHS.

Using the general procedures and methods described in the previousexamples, FIG. 23 is a flow chart illustrating the procedure fortransient transfection of COS-7 cells, in which cells in serum-freemedium were incubated in plasmid DNA in lipofectAMINE for 4 hours, serumwas added, and the cells were incubated for a further 40 hours. Thecells were then either incubated in regular medium containing serum fora further 48 hours before analysis (i.e. no further treatment), deprivedof serum for 48 hours to induce apoptosis before analysis, or treatedwith actinomycin D for 48 hours to induce apoptosis before analysis.

FIG. 22 is a Western blot illustrating transient expression of foreignproteins in COS-7 cells following transfection with pHM6. Protein wasisolated from COS-7 cells 48 hours after either mock transfection, ortransfection with pHM6-LacZ, pHM6-Antisense 3′ rF5A (pHM6-Antisense 3′UTR rat apoptosis eIF-5A), or pHM6-Sense rF5A (pHM6-Full length ratapoptosis eIF-5A). Five μg of protein from each sample was fractionatedby SDS-PAGE, transferred to a PVDF membrane, and Western blotted withanti-[HA]-peroxidase. The bound antibody was detected bychemiluminescence and exposed to x-ray film for 30 seconds. Expressionof LacZ (lane 2) and of sense rat apoptosis eIF-5A (lane 4) is clearlyvisible.

As described above, COS-7 cells were either mock transfected ortransfected with pHM6-Sense rF5A (pHM6-Full length rat eIF-5A). Fortyhours after transfection, the cells were induced to undergo apoptosis bywithdrawal of serum for 48 hours. The caspase proteolytic activity inthe transfected cell extract was measured using a fluorometrichomogenous caspase assay kit (Roche Diagnostics). DNA fragmentation wasalso measured using the FragEL DNA Fragmentation Apoptosis Detection kit(Oncogene) which labels the exposed 3′-OH ends of DNA fragments withfluorescein-labeled deoxynucleotides.

Additional COS-7 cells were either mock transfected or transfected withpHM6-Sense rF5A (pHM6-Full length rat eIF-5A). Forty hours aftertransfection, the cells were either grown for an additional 48 hours inregular medium containing serum (no further treatment), induced toundergo apoptosis by withdrawal of serum for 48 hours or induced toundergo apoptosis by treatment with 0.5 μg/ml of Actinomycin D for 48hours. The cells were either stained with Hoescht 33258, which depictsnuclear fragmentation accompanying apoptosis, or stained with AnnexinV-Cy3, which depicts phosphatidylserine exposure accompanying apoptosis.Stained cells were also viewed by fluorescence microscopy using a greenfilter and counted to determine the percentage of cells undergoingapoptosis. The total cell population was counted under visible light.

FIG. 25 illustrates enhanced apoptosis as reflected by increased caspaseactivity when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a60% increase in caspase activity.

FIG. 26 illustrates enhanced apoptosis as reflected by increased DNAfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a273% increase in DNA fragmentation. FIG. 27 illustrates detection ofapoptosis as reflected by increased nuclear fragmentation when COS-7cells were transiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. There is a greaterincidence of fragmented nuclei in cells expressing rat apoptosis-inducedeIF-5A. FIG. 28 illustrates enhanced apoptosis as reflected by increasednuclear fragmentation when COS-7 cells were transiently transfected withpHM6 containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a27% and 63% increase in nuclear fragmentation over control in non-serumstarved and serum starved samples, respectively.

FIG. 29 illustrates detection of apoptosis as reflected byphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-inducedeIF-5A in the sense orientation. FIG. 30 illustrates enhanced apoptosisas reflected by increased phosphatidylserine exposure when COS-7 cellswere transiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. Expression of ratapoptosis-induced eIF-5A resulted in a 140% and 198% increase inphosphatidylserine exposure over control, in non-serum starved and serumstarved samples, respectively.

FIG. 31 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a115% and 62% increase in nuclear fragmentation over control in untreatedand treated samples, respectively. FIG. 32 illustrates a comparison ofenhanced apoptosis under conditions in which COS-7 cells transientlytransfected with pHM6 containing full-length rat apoptosis-inducedeIF-5A in the sense orientation were either given no further treatmentor treatment to induce apoptosis.

Example 4

The present example demonstrates modulation of apoptotic activityfollowing administration of apoptosis factor 5A and DHS.

COS-7 cells were either mock transfected, transfected with pHM6-LacZ ortransfected with pHM6-Sense rF5A (pHM6-Full length rat eIF-5A) andincubated for 40 hours. Five μg samples of protein extract from eachsample were fractionated by SDS-PAGE, transferred to a PVDF membrane,and Western blotted with a monoclonal antibody that recognizes Bcl-2.Rabbit anti-mouse IgG conjugated to peroxidase was used as a secondaryantibody, and bound antibody was detected by chemiluminescence andexposure to x-ray film. Results are shown in FIG. 32. Less Bcl-2 isdetectable in cells transfected with pHM6-Sense rF5A than in thosetransfected with pHM6-LacZ; therefore, Bcl-2 is down-regulated.

Additional COS-7 cells were either mock transfected, transfected withpHM6-antisense 3′ rF5A (pHM6-antisense 3′ UTR of rat apoptosis-specificeIF-5A) or transfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A). Forty hours after transfection, the cellswere induced to undergo apoptosis by withdrawal of serum for 48 hours.Five μg samples of protein extract from each sample were fractionated bySDS-PAGE, transferred to a PVDF membrane, and Western blotted with amonoclonal antibody that recognizes Bcl-2. Rabbit anti-mouse IgGconjugated to peroxidase was used as a secondary antibody, and boundantibody was detected by chemiluminescence and exposure to x-ray film.

Also additionally, COS-7 cells were either mock transfected, transfectedwith pHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length rateIF-5A) and incubated for 40 hours. Five μg samples of protein extractfrom each sample were fractionated by SDS-PAGE, transferred to a PVDFmembrane, and Western blotted with a monoclonal antibody that recognizesp53. Goat anti-mouse IgG conjugated to alkaline phosphatase was used asa secondary antibody, and bound antibody was detected colorimetrically.

Finally, COS-7 cells were either mock transfected, transfected withpHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length rateIF-5A) and incubated for 40 hours. Five μg samples of protein extractfrom each sample were fractionated by SDS-PAGE, transferred to a PVDFmembrane, and probed with a monoclonal antibody that recognizes p53.Corresponding protein blots were probed with anti-[HA]-peroxidase todetermine the level of rat apoptosis-specific eIF-5A expression. Goatanti-mouse IgG conjugated to alkaline phosphatase was used as asecondary antibody, and bound antibody was detected bychemiluminescence.

FIG. 33 illustrates downregulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. Less Bcl-2 is detectable incells transfected with pHM6-Sense rF5A than in those transfected withpHM6-LacZ.

FIG. 34 illustrates upregulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the antisense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More Bcl-2 is detectable incells transfected with pHM6-antisense 3′ rF5A than in those mocktransfected or transfected with pHM6-Sense rF5A.

FIG. 35 illustrates upregulation of c-Myc when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More c-Myc is detectable incells transfected with pHM6-Sense rF5A than in those transfected withpHM6-LacZ or the mock control.

FIG. 36 illustrates upregulation of p53 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More p53 is detectable incells transfected with pHM6-Sense rF5A than in those transfected withpHM6-LacZ or the mock control.

FIG. 37 illustrates the dependence of p53 upregulation upon theexpression of pHM6-full length rat apoptosis-induced eIF-5A in COS-7cells. In the Western blot probed with anti-[HA]-peroxidase, the upperpanel illustrates the Coomassie-blue-stained protein blot and the lowerpanel illustrates the corresponding Western blot. More ratapoptosis-induced eIF-5A is detectable in the first transfection than inthe second transfection. In the Western blot probed with anti-p53, theupper panel in A illustrates a corresponding Coomassie-blue-stainedprotein blot and the lower panel illustrates the Western blot with p53.For the first transfection, more p53 is detectable in cells transfectedwith pHM6-Sense rF5A than in those transfected with pHM6-LacZ or themock control. For the second transfection in which there was lessexpression of rat apoptosis-induced eIF-5A, there was no detectabledifference in levels of p53 between cells transfected with pHM6-SenserF5A, pHM6-LacZ or the mock control.

Example 5

FIG. 47 depicts an experiment run on heart tissue to mimic the beatingof a human heart and the subsequent induced heart attack. FIG. 49 showsthe laboratory bench set up. A slice of human heart tissue removedduring valve replacement surgery to was hooked up to electrodes. A smallweight was attached to the heart tissue for ease in measuring thestrength of the heart beats. The electrodes provided an electricalstimulus to get the tissue to start beating. The levels of geneexpression for both apoptosis-specific eIF-5A (eIF-5a) and proliferatingeIF-5A (eIF-5b) were measured in the heart tissue before ischemia wasinduced. See FIG. 46. In the pre-ischemic heart tissue low levels ofboth eIF-5a and eIF-5b were produced and their levels were in relativebalance. During this time, oxygen and carbon dioxide were delivered in abuffer to the heart at 92.5% and 7.5%, respectively. Later, the oxygenlevel was reduced and the nitrogen level was increased, to induceischemia and finally a “heart attack.” The heart tissue stopped beating.The oxygen levels were then returned to normal, the heart tissue waspulsed again with an electrical stimulus to start the heart beatingagain. After the “heart attack” the expression levels ofapoptosis-specific eIF-5a and proliferating eIF-5A (eIF-5b) were againmeasured. This time, there was a significant increase in the level ofexpression of the apoptosis-specific eIF-5A levels, whereas the increasein the level of expression of proliferating eIF-5A (eIF-5b) wasnoticeably less. See FIG. 46.

After the “heart attack” the heart did not beat as strongly, asindicated by less compression/movement of the attached weight, thusindicating that the heart tissue cells were being killed rapidly due tothe presence of apoptosis-specific eIF-5A.

The EKG results are depicted in FIG. 48. On the left side of the panelsa normal heart beat is shown (the pre-ischemic heart tissue). After the“heart attack” (straight line), and the re-initiation of the heart beat,the EKG shows decreased activity due to muscle cell death. The EKG showsrelative loss in strength of heart beat.

Example 6 Human Cell Line Culture Conditions

Human Lamina Cribrosa and Astrocyte Culture

Paired human eyes were obtained within 48 hours post mortem from the EyeBank of Canada, Ontario Division. Optic nerve heads (with attached pole)were removed and placed in Dulbecco's modified Eagle's medium (DMEM)supplemented with antibiotic/antimycotic, glutamine, and 10% FBS for 3hours. The optic nerve head (ONH) button was retrieved from each tissuesample and minced with fine dissecting scissors into four small pieces.Explants were cultured in 12.5 cm² plastic culture flasks in DMEMmedium. Growth was observed within one month in viable explants. Oncethe cells reached 90% confluence, they were trypsinized and subjected todifferential subculturing to produce lamina cribrosa (LC) and astrocytecell populations. Specifically, LC cells were subcultured in 25 cm²flasks in DMEM supplemented with gentamycin, glutamine, and 10% FBS,whereas astrocytes were expanded in 25 cm² flasks containing EBMcomplete medium (Clonetics) with no FBS. FBS was added to astrocytecultures following 10 days of subculture. Cells were maintained andsubcultured as per this protocol.

Cell populations obtained by differential subculturing werecharacterized for identity and population purity using differentialfluorescent antibody staining on 8 well culture slides. Cells were fixedin 10% formalin solution and washed three times with Dulbecco'sPhosphate Buffered Saline (DPBS). Following blocking with 2% nonfat milkin DPBS, antibodies were diluted in 1% BSA in DPBS and applied to thecells in 6 of the wells. The remaining two wells were treated with only1% bovine serum albumin (BSA) solution and no primary antibody ascontrols. Cells were incubated with the primary antibodies for one hourat room temperature and then washed three times with DPBS. Appropriatesecondary antibodies were diluted in 1% BSA in DPBS, added to each welland incubated for 1 hour. Following washing with DPBS, the chambersseparating the wells of the culture slide were removed from the slide,and the slide was immersed in double distilled water and then allowed toair-dry. Fluoromount (Vector Laboratories) was applied to each slide andoverlayed by 22×60 mm coverglass slips.

Immunofluorescent staining was viewed under a fluorescent microscopewith appropriate filters and compared to the control wells that were nottreated with primary antibody. All primary antibodies were obtained fromSigma unless otherwise stated. All secondary antibodies were purchasedfrom Molecular Probes. Primary antibodies used to identify LC cellswere: anti-collagen I, anti-collagen IV, anti-laminin, anti-cellularfibronectin. Primary antibodies used to identify astrocytes were:anti-galactocerebroside (Chemicon International), anti-A2B5 (ChemiconInternational), anti-NCAM, anti-human Von willebrand Factor. Additionalantibodies used for both cell populations included anti-glial fibrillary(GFAP) and anti-alpha-smooth muscle actin. Cell populations weredetermined to be comprised of LC cells if they stained positively forcollagen I, collagen IV, laminin, cellular fibronectin, alpha smoothmuscle actin and negatively for glial fibrillary (GFAP). Cellpopulations were determined to be comprised of astrocytes if theystained positively for NCAM, glial fibrillary (GFAP), and negatively forgalactocerebroside, A2B5, human Von willebrand Factor, and alpha smoothmuscle actin.

In this preliminary study, three sets of human eyes were used toinitiate cultures. LC cell lines #506, #517, and #524 were establishedfrom the optic nerve heads of an 83-year old male, a 17-year old male,and a 26-year old female, respectively. All LC cell lines have beenfully characterized and found to contain greater than 90% LC cells.

RKO Cell Culture

RKO (American Type Culture Collection CRL-2577), a human colon carcinomacell line expressing wild-type p53, was used to test the antisenseoligonucleotides for the ability to suppress eIF-5A1 protein expression.RKO were cultured in Minimum Essential Medium Eagle (MEM) withnon-essential amino acids, Earle's salts, and L-glutamine. The culturemedia was supplemented with 10% fetal bovine serum (FBS) and 100 unitsof penicillin/streptomycin. The cells were grown at 37° C. in ahumidified environment of 5% CO₂ and 95% air. The cells were subculturedevery 3 to 4 days by detaching the adherent cells with a solution of0.25% trypsin and 1 mM EDTA. The detached cells were dispensed at asplit ratio of 1:10 to 1:12 into a new culture dish with fresh media.

HepG2 Cell Culture

HepG2, a human hepatocellular carcinoma cell line, was used to test theability of an antisense oligo directed against human eIF-5A1 to blockproduction of TNF-α in response to treatment with IL-1β. HepG2 cellswere cultured in DMEM supplemented with gentamycin, glutamine, and 10%FBS and grown at 37° C. in a humidified environment of 5% CO₂ and 95%air.

Example 7 Induction of Apoptosis

Apoptosis was induced in RKO and lamina cribrosa cells using ActinomycinD, an RNA polymerase inhibitor, and camptothecin, a topoisomeraseinhibitor, respectively. Actinomycin D was used at a concentration of0.25 μg/ml and camptothecin was used at a concentration of 20, 40, or 50Apoptosis was also induced in lamina cribrosa cells using a combinationof camptothecin (50 μM) and TNF-α (10 ng/ml). The combination ofcamptothecin and TNF-α was found to be more effective at inducingapoptosis than either camptothecin or TNF-α alone.

Antisense Oligonucleotides

A set of three antisense oligonucleotides targeted against human eIF-5A1were designed by, and purchased from, Molecula Research Labs. Thesequence of the first antisense oligonucleotide targeted against humaneIF-5A1 (#1) was 5′ CCT GTC TCG AAG TCC AAG TC 3′ (SEQ ID NO: 63). Thesequence of the second antisense oligonucleotide targeted against humaneIF-5A1 (#2) was 5′ GGA CCT TGG CGT GGC CGT GC 3′ (SEQ ID NO: 64). Thesequence of the third antisense oligonucleotide targeted against humaneIF-5A1 (#3) was 5′ CTC GTA CCT CCC CGC TCT CC 3′ (SEQ ID NO: 65). Thecontrol oligonucleotide had the sequence 5′ CGT ACC GGT ACG GTT CCA GG3′ (SEQ ID NO: 66). A fluorescein isothiocyanate (FITC)-labeledantisense oligonucleotide (Molecula Research Labs) was used to monitortransfection efficiency and had the sequence 5′ GGA CCT TGG CGT GGC CGTGCX 3′ (SEQ ID NO: 67), where X is the FITC label. All antisenseoligonucleotides were fully phosphorothioated.

Transfection of Antisense Oligonucleotides

The ability of the eIF-5A1 antisense oligonucleotides to block eIF-5A1protein expression was tested in RKO cells. RKO cells were transfectedwith antisense oligonucleotides using the transfection reagent,Oligofectamine (Invitrogen). Twenty four hours prior to transfection,the cells were split onto a 24 well plate at 157,000 per well in MEMmedia supplemented with 10% FBS but lacking penicillin/streptomycin.Twenty four hours later the cells had generally reached a confluency ofapproximately 50%. RKO cells were either mock transfected, ortransfected with 100 nM or 200 nM antisense oligonucleotide.Transfection medium sufficient for one well of an 24 well plate wasprepared by diluting 0, 1.25, or 2.5 μl of a 20 μM stock of antisenseoligonucleotide with serum-free MEM to a final volume of 42.5 μl andincubating the mixture at room temperature for 15 minutes. 1.5 μl ofOligofectamine was diluted in 6 μl of serum-free MEM and incubated for7.5 minutes at room temperature. After 5 minutes the dilutedOligofectamine mixture was added to the DNA mixture and incubatedtogether at room temperature for 20 minutes. The cells were washed oncewith serum-free MEM before adding 200 μl of MEM to the cells andoverlaying 50 μl of transfection medium. The cells were placed back inthe growth chamber for 4 hours. After the incubation, 125 μl of MEM+30%FBS was added to the cells. The cells were then cultured for a further48 hours, treated with 0.25 μg/ml Actinomycin D for 24 hours, and thencell extract was harvested for Western blot analysis.

Transfection of lamina cribrosa cells was also tested using 100 and 200nM antisense oligonucleotide and Oligofectamine using the same proceduredescribed for RKO cells. However, effective transfection of laminacribrosa cells was achieved by simply adding antisense oligonucleotide,diluted from 1 μM to 10 μM in serum-free media, to the cells for 24hours and thereafter replacing the media with fresh antisenseoligonucleotides diluted in serum-containing media every 24 hours for atotal of two to five days.

The efficiency of antisense oligonucleotide transfection was optimizedand monitored by performing transfections with an FITC-labeled antisenseoligonucleotide having the same sequence as eIF-5A1 antisenseoligonucleotide #2 but conjugated to FITC at the 3′ end. RKO and laminacribrosa cells were transfected with the FITC-labeled antisenseoligonucleotide on an 8-well culture slide. Forty-eight hours later thecells were washed with PBS and fixed for 10 minutes in 3.7% formaldehydein PBS. The wells were removed and mounting media (Vectashield) wasadded, followed by a coverslip. The cells were then visualized under UVlight on a fluorescent microscope nucleus using a fluorescein filter(Green H546, filter set 48915) and cells fluorescing bright green weredetermined to have taken up the oligonucleotide.

Detection of Apoptosis

Following transfection of lamina cribosa cells with antisenseoligonucleotides and induction of apoptosis with camptothecin, thepercentage of cells undergoing apoptosis in cells treated with eithercontrol antisense oligonucleotide or antisense oligonucleotide eIF-5A1SEQ ID NO:26 was determined. Two methods were used to detect apoptoticlamina cribosa cells—Hoescht staining and DeadEnd™ Fluorometric TUNEL.The nuclear stain, Hoescht, was used to label the nuclei of laminacribosa cells in order to identify apoptotic cells based onmorphological features such as nuclear fragmentation and condensation. Afixative, consisting of a 3:1 mixture of absolute methanol and glacialacetic acid, was prepared immediately before use. An equal volume offixative was added to the media of cells growing on a culture slide andincubated for 2 minutes. The media/fixative mixture was removed from thecells and discarded and 1 ml of fixative was added to the cells. After 5minutes the fixative was discarded and 1 ml of fresh fixative was addedto the cells and incubated for 5 minutes. The fixative was discarded andthe cells were air-dried for 4 minutes before adding 1 ml of Hoeschtstain (0.5 μg/ml Hoescht 33258 in PBS). After a 10 minute incubation inthe dark, the staining solution was discarded, the chambers separatingthe wells of the culture slide were removed, and the slide was washed 3times for 1 minute with deionized water. After washing, a few drops ofMcIlvaine's buffer (0.021 M citric acid, 0.058 M Na₂HPO₄.7H₂O; pH 5.6)was added to the cells and overlaid with a coverslip. The stained cellswere viewed under a fluorescent microscope using a UV filter. Cells withbrightly stained or fragmented nuclei were scored as apoptotic. Aminimum of 200 cells were counted per well.

The DeadEnd™ Fluorometric TUNEL (Promega) was used to detect the DNAfragmentation that is a characteristic feature of apoptotic cells.Following Hoescht staining, the culture slide was washed briefly withdistilled water, and further washed by immersing the slide twice for 5minutes in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mMNa₂HPO₄), blotting the slide on paper towel between washes. The cellswere permeabilized by immersing them in 0.2% Triton X-100 in PBS for 5minutes. The cells were then washed again by immersing the slide twicefor 5 minutes in PBS and blotting the slide on paper towel betweenwashes. 25 μl of equilibration buffer [200 mM potassium cacodylate (pH6.6), 25 mM Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg/ml bovineserum albumin, and 2.5 mM cobalt chloride] was added per well andincubated for 5 to 10 minutes. During equilibration, 30 μl of reactionmixture was prepared for each well by mixing in a ratio of 45:5:1,respectively, equilibration buffer, nucleotide mix [50 μMfluorescein-12-dUTP, 100 μM dATP, 10 mM Tris-HCl (pH 7.6), and 1 mMEDTA], and terminal deoxynucleotidyl transferase enzyme (Tdt, 25 U/μl).After the incubation in equilibration buffer, 30 μl of reaction mixturewas added per well and overlayed with a coverslip. The reaction wasallowed to proceed in the dark at 37° C. for 1 hour. The reaction wasterminated by immersing the slide in 2×SSC [0.3 M NaCl, and 30 mM sodiumcitrate (pH 7.0)] and incubating for 15 minutes. The slide was thenwashed by immersion in PBS three times for 5 minutes. The PBS wasremoved by sponging around the wells with a Kim wipe, a drop of mountingmedia (Oncogene research project, JA1750-4ML) was added to each well,and the slide was overlayed with a coverslip. The cells were viewedunder a fluorescent microscope using a UV filter (UV-G 365, filter set487902) in order to count the Hoescht-stained nuclei. Any cells withbrightly stained or fragmented nuclei were scored as apoptotic. Usingthe same field of view, the cells were then viewed using a fluoresceinfilter (Green H546, filter set 48915) and any nuclei fluorescing brightgreen were scored as apoptotic. The percentage of apoptotic cells in thefield of view was calculated by dividing the number of bright greennuclei counted using the fluorescein filter by the total number ofnuclei counted under the UV filter. A minimum of 200 cells were countedper well.

FIGS. 54-57 depict the results of these studies. The percentage ofapoptotic cells in samples having been transfected withapoptosis-specific eIF-5A1 is clearly much less than seen in cellshaving been transfected with the control oligonucleotide.

Protein Extraction and Western Blotting

Protein from transfected RKO cells was harvested for Western blotanalysis by washing the cells with PBS, adding 40 μl of hot lysis buffer[0.5% SDS, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 8.0)] per well. Thecells were scraped and the resulting extract was transferred to amicrofuge tube, boiled for 5 minutes, and stored at −20° C. The proteinwas quantitated using the Bio-Rad Protein Assay (Bio-Rad) according tothe manufacturer's instructions.

For Western blotting 5 μg of total protein was separated on a 12%SDS-polyacrylamide gel. The separated proteins were transferred to apolyvinylidene difluoride membrane. The membrane was then incubated forone hour in blocking solution (5% skim milk powder in PBS) and washedthree times for 15 minutes in 0.05% Tween-20/PBS. The membrane wasstored overnight in PBS-T at 4° C. After being warmed to roomtemperature the next day, the membrane was blocked for 30 seconds in 1μg/ml polyvinyl alcohol. The membrane was rinsed 5 times in deionizedwater and then blocked for 30 minutes in a solution of 5% milk in 0.025%Tween-20/PBS. The primary antibody was preincubated for 30 minutes in asolution of 5% milk in 0.025% Tween-20/PBS prior to incubation with themembrane.

Several primary antibodies were used. A monoclonal antibody fromOncogene which recognizes p53 (Ab-6) and a polyclonal antibody directedagainst a synthetic peptide (amino-CRLPEGDLGKEIEQKYD-carboxy) (SEQ IDNO:68) homologous to the c-terminal end of human eIF-5A1 that was raisedin chickens (Gallus Immunotech). An anti-β-actin antibody (Oncogene) wasalso used to demonstrate equal loading of protein. The monoclonalantibody to p53 was used at a dilution of 0.05 μg/ml, the antibodyagainst eIF-5A1 was used at a dilution of 1:1000, and the antibodyagainst actin was used at a dilution of 1:20,000. After incubation withprimary antibody for 60 to 90 minutes, the membrane was washed 3 timesfor 15 minutes in 0.05% Tween-20/PBS. Secondary antibody was thendiluted in 1% milk in 0.025% Tween-20/PBS and incubated with themembrane for 60 to 90 minutes. When p53 (Ab-6) was used as the primaryantibody, the secondary antibody used was a rabbit anti-mouse IgGconjugated to peroxidase (Sigma) at a dilution of 1:5000. Whenanti-eIF-5A1 was used as the primary antibody, a rabbit anti-chicken IgYconjugated to peroxidase (Gallus Immunotech) was used at a dilution of1:5000. The secondary antibody used with actin was a goat anti-mouse IgMconjugated to peroxidase (Calbiochem) used at a dilution of 1:5000.After incubation with the secondary antibody, the membrane was washed 3times in PBS-T.

The ECL Plus Western blotting detection kit (Amersham Pharmacia Biotech)was used to detect peroxidase-conjugated bound antibodies. In brief, themembrane was lightly blotted dry and then incubated in the dark with a40:1 mix of reagent A and reagent B for 5 minutes. The membrane wasblotted dry, placed between sheets of acetate, and exposed to X-ray filmfor time periods varying from 10 seconds to 30 minutes. The membrane wasstripped by submerging the membrane in stripping buffer [100 mM2-Mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7)], andincubating at 50° C. for 30 minutes. The membrane was then rinsed indeionized water and washed twice for 10 minutes in large volumes of0.05% Tween-20/PBS. Membranes were stripped and re-blotted up to threetimes.

Example 8 Construction of siRNA

Small inhibitory RNAs (siRNAs) directed against human eIF-5A1 were usedto specifically suppress expression of eIF-5A1 in RKO and laminacribrosa cells. Six siRNAs were generated by in vitro transcriptionusing the Silencer™ siRNA Construction Kit (Ambion Inc.). Four siRNAswere generated against human eIF-5A1 (siRNAs #1 to #4) (SEQ IDNO:30-33). See FIG. 70. Two siRNAs were used as controls; an siRNAdirected against GAPDH provided in the kit, and an siRNA (siRNA #5) (SEQID NO:34) which has the reverse sequence of the eIF-5A1-specific siRNA#1 (SEQ ID NO:30) but does not itself target eIF-5A1. The siRNAs weregenerated according to the manufacturer's protocol. In brief, DNAoligonucleotides encoding the desired siRNA strands were used astemplates for T7 RNA polymerase to generate individual strands of thesiRNA following annealing of a T7 promoter primer and a fill-in reactionwith Klenow fragment. Following transcription reactions for both thesense and antisense strands, the reactions were combined and the twosiRNA strands were annealed, treated with DNase and RNase, and thencolumn purified. The sequence of the DNA oligonucleotides (T7 primerannealing site underlined) used to generate the siRNAs were: siRNA #1antisense 5′ AAAGGAATGACTTCCAGCTGACCTGTCTC 3′ (SEQ ID NO: 69) and siRNA#1 sense 5′ AATCAGCTGGAAGTCATTCCTCCTGTCTC 3′ (SEQ ID NO: 70); siRNA #2antisense 5′ AAGATCGTCGAGATGTCTACTCCTGTCTC 3′ (SEQ ID NO: 71) and siRNA#2 sense 5′ AAAGTAGACATCTCGACGATCCCTGTCTC 3′ (SEQ ID NO: 72); siRNA #3antisense 5′ AAGGTCCATCTGGTTGGTATTCCTGTCTC 3′ (SEQ ID NO: 73) and siRNA#3 sense 5′ AAAATACCAACCAGATGGACCCCTGTCTC 3′ (SEQ ID NO: 74); siRNA #4antisense 5′ AAGCTGGACTCCTCCTACACACCTGTCTC 3′ (SEQ ID NO: 75) and siRNA#4 sense 5′ AATGTGTAGGAGGAGTCCAGCCCTGTCTC 3′ (SEQ ID NO: 76); siRNA #5antisense 5′ AAAGTCGACCTTCAGTAAGGACCTGTCTC 3′ (SEQ ID NO: 77) and siRNA#5 sense 5′ AATCCTTACTGAAGGTCGACTCCTGTCTC 3′ (SEQ ID NO: 78).

The Silencer™ siRNA Labeling Kit—FAM (Ambion) was used to label GAPDHsiRNA with FAM in order to monitor the uptake of siRNA into RKO andlamina cribrosa cells. After transfection on 8-well culture slides,cells were washed with PBS and fixed for 10 minutes in 3.7% formaldehydein PBS. The wells were removed and mounting media (Vectashield) wasadded, followed by a coverslip. Uptake of the FAM-labeled siRNA wasvisualized under a fluorescent microscope under UV light using afluorescein filter. The GAPDH siRNA was labeled according to themanufacturer's protocol.

Transfection of siRNA

RKO cells and lamina cribrosa cells were transfected with siRNA usingthe same transfection protocol. RKO cells were seeded the day beforetransfection onto 8-well culture slides or 24-well plates at a densityof 46,000 and 105,800 cells per well, respectively. Lamina cribrosacells were transfected when cell confluence was at 40 to 70% and weregenerally seeded onto 8-well culture slides at 7500 to 10,000 cells perwell three days prior to transfection. Transfection medium sufficientfor one well of an 8-well culture slide was prepared by diluting 25.5pmoles of siRNA stock to a final volume of 21.2 μl in Opti-Mem (Sigma).0.425 μl of Lipofectamine 2000 was diluted to a final volume of 21.2 μlin Opti-Mem and incubated for 7 to 10 minutes at room temperature. Thediluted Lipofectamine 2000 mixture was then added to the diluted siRNAmixture and incubated together at room temperature for 20 to 30 minutes.The cells were washed once with serum-free media before adding 135 μl ofserum-free media to the cells and overlaying the 42.4 μl of transfectionmedium. The cells were placed back in the growth chamber for 4 hours.After the incubation, 65 μl of serum-free media+30% FBS was added to thecells. Transfection of siRNA into cells to be used for Western blotanalysis were performed in 24-well plates using the same conditions asthe transfections in 8-well slides except that the volumes wereincreased by 2.3 fold.

Following transfection, RKO and lamina cribrosa cells were incubated for72 hours prior to collection of cellular extract for Western blotanalysis. In order to determine the effectiveness of the siRNAs directedagainst eIF-5A1 to block apoptosis, lamina cribrosa cells were treatedwith 50 μM of camptothecin (Sigma) and 10 ng/ml of TNF-α (LeincoTechnologies) to induce apoptosis either 48 or 72 hours aftertransfection. The cells were stained with Hoescht either 24 or 48 hourslater in order to determine the percentage of cells undergoingapoptosis.

Example 9 Detection of Apoptosis

Following transfection of lamina cribrosa cells with antisenseoligonucleotides and induction of apoptosis with camptothecin, thepercentage of cells undergoing apoptosis in cells treated with eithercontrol antisense oligonucleotide or antisense oligonucleotide eIF-5A1#2 was determined. Two methods were used to detect apoptotic laminacribrosa cells—Hoescht staining and DeadEnd™ Fluorometric TUNEL. Thenuclear stain, Hoescht, was used to label the nuclei of lamina cribrosacells in order to identify apoptotic cells based on morphologicalfeatures such as nuclear fragmentation and condensation. A fixative,consisting of a 3:1 mixture of absolute methanol and glacial aceticacid, was prepared immediately before use. An equal volume of fixativewas added to the media of cells growing on a culture slide and incubatedfor 2 minutes. The media/fixative mixture was removed from the cells anddiscarded and 1 ml of fixative was added to the cells. After 5 minutesthe fixative was discarded and 1 ml of fresh fixative was added to thecells and incubated for 5 minutes. The fixative was discarded and thecells were air-dried for 4 minutes before adding 1 ml of Hoescht stain(0.5 μg/ml Hoescht 33258 in PBS). After a 10 minute incubation in thedark, the staining solution was discarded, the chambers separating thewells of the culture slide were removed, and the slide was washed 3times for 1 minute with deionized water. After washing, a few drops ofMcIlvaine's buffer (0.021 M citric acid, 0.058 M Na₂HPO₄.7H₂O; pH 5.6)was added to the cells and overlaid with a coverslip. The stained cellswere viewed under a fluorescent microscope using a UV filter. Cells withbrightly stained or fragmented nuclei were scored as apoptotic. Aminimum of 200 cells were counted per well.

The DeadEnd™ Fluorometric TUNEL (Promega) was used to detect the DNAfragmentation that is a characteristic feature of apoptotic cells.Following Hoescht staining, the culture slide was washed briefly withdistilled water, and further washed by immersing the slide twice for 5minutes in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mMNa₂HPO₄), blotting the slide on paper towel between washes. The cellswere permeabilized by immersing them in 0.2% Triton X-100 in PBS for 5minutes. The cells were then washed again by immersing the slide twicefor 5 minutes in PBS and blotting the slide on paper towel betweenwashes. 25 μl of equilibration buffer [200 mM potassium cacodylate (pH6.6), 25 mM Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg/ml bovineserum albumin, and 2.5 mM cobalt chloride] was added per well andincubated for 5 to 10 minutes. During equilibration, 30 μl of reactionmixture was prepared for each well by mixing in a ratio of 45:5:1,respectively, equilibration buffer, nucleotide mix [50 μMfluorescein-12-dUTP, 100 μM dATP, 10 mM Tris-HCl (pH 7.6), and 1 mMEDTA], and terminal deoxynucleotidyl transferase enzyme (Tdt, 25 U/μl).After the incubation in equilibration buffer, 30 μl of reaction mixturewas added per well and overlayed with a coverslip. The reaction wasallowed to proceed in the dark at 37° C. for 1 hour. The reaction wasterminated by immersing the slide in 2×SSC [0.3 M NaCl, and 30 mM sodiumcitrate (pH 7.0)] and incubating for 15 minutes. The slide was thenwashed by immersion in PBS three times for 5 minutes. The PBS wasremoved by sponging around the wells with a Kim wipe, a drop of mountingmedia (Oncogene research project, JA1750-4ML) was added to each well,and the slide was overlayed with a coverslip. The cells were viewedunder a fluorescent microscope using a UV filter (UV-G 365, filter set487902) in order to count the Hoescht-stained nuclei. Any cells withbrightly stained or fragmented nuclei were scored as apoptotic. Usingthe same field of view, the cells were then viewed using a fluoresceinfilter (Green H546, filter set 48915) and any nuclei fluorescing brightgreen were scored as apoptotic. The percentage of apoptotic cells in thefield of view was calculated by dividing the number of bright greennuclei counted using the fluorescein filter by the total number ofnuclei counted under the UV filter. A minimum of 200 cells were countedper well.

Protein Extraction and Western Blotting

Protein from transfected RKO cells was harvested for Western blotanalysis by washing the cells with PBS, adding 40 μl of hot lysis buffer[0.5% SDS, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 8.0)] per well. Thecells were scraped and the resulting extract was transferred to aneppendorf, boiled for 5 minutes, and stored at −20° C. The protein wasquantitated using the Bio-Rad Protein Assay (Bio-Rad) according to themanufacturer's instructions.

For Western blotting 5 μg of total protein was separated on a 12%SDS-polyacrylamide gel. The separated proteins were transferred to apolyvinylidene difluoride membrane. The membrane was then incubated forone hour in blocking solution (5% skim milk powder in PBS) and washedthree times for 15 minutes in 0.05% Tween-20/PBS. The membrane wasstored overnight in PBS-T at 4° C. After being warmed to roomtemperature the next day, the membrane was blocked for 30 seconds in 1μg/ml polyvinyl alcohol. The membrane was rinsed 5 times in deionizedwater and then blocked for 30 minutes in a solution of 5% milk in 0.025%Tween-20/PBS. The primary antibody was preincubated for 30 minutes in asolution of 5% milk in 0.025% Tween-20/PBS prior to incubation with themembrane.

Several primary antibodies were used. A monoclonal antibody fromOncogene which recognizes p53 (Ab-6; Oncogene), a monoclonal whichrecognizes human bcl-2 (Oncogene), and a polyclonal antibody directedagainst a synthetic peptide (amino-CRLPEGDLGKEIEQKYD-carboxy) (SEQ IDNO: 68) homologous to the c-terminal end of human eIF-5A1 that wasraised in chickens (Gallus Immunotech). An anti-β-actin antibody(Oncogene) was also used to demonstrate equal loading of protein. Themonoclonal antibody to p53 was used at a dilution of 0.05 μg/ml, theantibody against bcl-2 was used at a dilution of 1:3500, the antibodyagainst eIF-5A1 was used at a dilution of 1:1000, and the antibodyagainst actin was used at a dilution of 1:20,000. After incubation withprimary antibody for 60 to 90 minutes, the membrane was washed 3 timesfor 15 minutes in 0.05% Tween-20/PBS. Secondary antibody was thendiluted in 1% milk in 0.025% Tween-20/PBS and incubated with themembrane for 60 to 90 minutes. When p53 (Ab-6) was used as the primaryantibody, the secondary antibody used was a rabbit anti-mouse IgGconjugated to peroxidase (Sigma) at a dilution of 1:5000. Whenanti-eIF-5A1 was used as the primary antibody, a rabbit anti-chicken IgYconjugated to peroxidase (Gallus Immunotech) was used at a dilution of1:5000. The secondary antibody used with actin was a goat anti-mouse IgMconjugated to peroxidase (Calbiochem) used at a dilution of 1:5000.After incubation with the secondary antibody, the membrane was washed 3times in PBS-T.

The ECL Plus Western blotting detection kit (Amersham Pharmacia Biotech)was used to detect peroxidase-conjugated bound antibodies. In brief, themembrane was lightly blotted dry and then incubated in the dark with a40:1 mix of reagent A and reagent B for 5 minutes. The membrane wasblotted dry, placed between sheets of acetate, and exposed to X-ray filmfor time periods varying from 10 seconds to 30 minutes. The membrane wasstripped by submerging the membrane in stripping buffer [100 mM2-Mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7)], andincubating at 50° C. for 30 minutes. The membrane was then rinsed indeionized water and washed twice for 10 minutes in large volumes of0.05% Tween-20/PBS. Membranes were stripped and re-probed up to threetimes.

Example 10 Quantification of HepG2 TNF-α Production

HepG2 cells were plated at 20,000 cells per well onto 48-well plates.Seventy two hours later the media was removed and fresh media containingeither 2.5 μM control antisense oligonucleotide or 2.5 μM antisenseoligonucleotide eIF-5A1 #2 (SEQ ID NO:26) was added to the cells. Freshmedia containing antisense oligonucleotides was added after twenty fourhours. After a total of 48 hours incubation with the oligonucleotides,the media was replaced with media containing interleukin 1β (IL-1β, 1000pg/ml; Leinco Technologies) and incubated for 6 hours. The media wascollected and frozen (−20° C.) for TNF-α quantification. Additionalparallel incubations with untreated cells (without antisenseoligonucleotide and IL-1β) and cells treated with only IL-1β were usedfor controls. All treatments were done in duplicate. TNF-α released intothe media was measured by ELISA assays (Assay Designs Inc.) according tothe manufacturer's protocol.

Example 11

The following experiments show that antisense apoptosis factor 5Anucleotides were able to inhibit expression of apoptosis factor 5A aswell as p53. RKO cells were either left untransfected, mock transfected,or transfected with 200 nM of antisense oligonucleotides eIF-5A1 #1, #2,or #3 (SEQ ID NO: 25, 26, and 27)/RKO cells were also transfected with100 nM of antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26).Forty-eight hours after transfection, the cells were treated with 0.25μg/ml Actinomycin D. Twenty-four hours later, the cell extract washarvested and 5 μg of protein from each sample was separated on anSDS-PAGE gel, transferred to a PVDF membrane, and Western blotted withan antibody against eIF-5A1. After chemiluminescent detection, themembrane was stripped and reprobed with an antibody against p53. Afterchemiluminescent detection, the membrane was stripped again and reprobedwith an antibody against actin. See FIG. 52 which shows the levels ofprotein produced by RKO cells after being treated with antisense oligo1, 2 and 3 (to apoptosis factor 5A) (SEQ ID NO:25,26, and 27,respectively). The RKO cells produced less apoptosis factor 5A as wellas less p53 after having been transfected with the antisense apoptosisfactor 5A nucleotides.

Example 12

The following experiments show that antisense apoptosis factor 5Anucleotides were able to reduce apoptosis.

In one experiment, the lamina cribrosa cell line #506 was either (A)transfected with 100 nM of FITC-labeled antisense oligonucleotide usingOligofectamine transfection reagent or (B) transfected with 10 μM ofnaked FITC-labeled antisense oligonucleotide diluted directly inserum-free media. After 24 hours fresh media containing 10% FBS andfresh antisense oligonucleotide diluted to 10 μM was added to the cells.The cells, (A) and (B), were fixed after a total of 48 hours andvisualized on a fluorescent microscope under UV light using afluorescein filter. FIG. 53 shows uptake of the fluorescently labeledantisense oligonucleotide.

In another experiment, the lamina cribrosa cell line #506 wastransfected with 10 μM of either the control antisense oligonucleotideor antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26) for a total of 4days. Forty-eight hours after beginning antisense oligonucleotidetreatment, the cells were treated with either 20 μM or 40 μMcamptothecin for 48 hours. Antisense oligonucleotide andcamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht andTUNEL. See FIG. 54.

In another experiment, the lamina cribrosa cell line #506 wastransfected with 10 μM of either the control antisense oligonucleotideor antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26). Twenty-fourhours later the media was changed and fresh antisense oligonucleotideswere added. Forty-eight hours after beginning antisense oligonucleotidetreatment, the antisense-oligonucleotides were removed and the cellswere treated with 20 μM camptothecin for 3 days. Thecamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht andTUNEL. See FIG. 55.

In yet another experiment, the lamina cribrosa cell line #517 wastransfected with 1 μM of either the control antisense oligonucleotide orantisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26) for a total of fivedays. Forty-eight hours after beginning antisense oligonucleotidetreatment, the cells were treated with 20 μM camptothecin for either 3or 4 days. Antisense oligonucleotide and camptothecin-containing mediawas changed daily. The percentage of apoptotic cells was determined bylabeling the cells with Hoescht and TUNEL. See FIG. 56.

In another experiment, the lamina cribrosa cell line #517 wastransfected with 2.5 μM of either the control antisense oligonucleotideor antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26) for a total offive days. Forty-eight hours after beginning antisense oligonucleotidetreatment, the cells were treated with 40 μM camptothecin for 3 days.Antisense oligonucleotide and camptothecin-containing media was changeddaily. The percentage of apoptotic cells was determined by labeling thecells with Hoescht. See FIG. 57.

In another experiment, the lamina cribrosa cell line #517 wastransfected with either 1 μM or 2.5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26)for a total of five days. Forty-eight hours after beginning antisenseoligonucleotide treatment, the cells were treated with 40 μMcamptothecin for 3 days. Antisense oligonucleotide andcamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht. SeeFIG. 58.

In another experiment, the lamina cribrosa cell line #517 was lefteither untreated, or was treated with 10 ng/ml TNF-α, 50 μMcamptothecin, or 10 ng/ml TNF-α and 50 μM camptothecin. The percentageof apoptotic cells was determined by labeling the cells with Hoescht.See FIG. 59.

In another experiment, the lamina cribrosa cell lines #506 and #517 weretransfected with either 2.5 μM or 5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26)for a total of two days. Fresh media containing antisenseoligonucleotides was added after 24 hours. Forty-eight hours afterbeginning antisense oligonucleotide treatment, the cells were treatedwith 50 μM camptothecin and 10 ng/ml TNF-α for 2 days. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht. SeeFIG. 60.

In another experiment, the lamina cribrosa cell lines #506, #517, and#524 were transfected with 2.5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide eIF-5A1 #2 (SEQ ID NO:26)for a total of two days. Fresh media containing antisenseoligonucleotides was added after 24 hours. Forty-eight hours afterbeginning antisense oligonucleotide treatment, the cells were treatedwith 50 μM camptothecin and 10 ng/ml TNF-α for 2 days. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht. SeeFIG. 61.

Example 13

The following experiments show that cells transfected with siRNAstargeted against apoptosis factor 5A expressed less apoptosis factor 5A.The experiments also show that siRNAs targeted against apoptosis factor5A were able to reduce apoptosis.

In one experiment, the lamina cribrosa cell line #517 was transfectedwith 100 nM of FAM-labeled siRNA using Lipofectamine 2000 transfectionreagent either with serum (A) or without serum (B) during transfection.The cells, (A) and (B), were fixed after a total of 24 hours andvisualized on a fluorescent microscope under UV light using afluorescein filter. See FIG. 62.

In another experiment, RKO cells were transfected with 100 nM of siRNAeither in the presence or absence of serum during the transfection. SixsiRNAs were transfected, two control siRNAs (siRNA #5 (SEQ ID NO: 34)and one targeted against GAPDH) and four targeted against eIF-5A1 (siRNA#1 to #4) (SEQ ID NO:30-34). Seventy-two hours after transfection, thecell extract was harvested and 5 μg of protein from each sample wasseparated on an SDS-PAGE gel, transferred to a PVDF membrane, andWestern blotted with an antibody against eIF-5A1. After chemiluminescentdetection, the membrane was stripped and re-probed with an antibodyagainst bcl-2. After chemiluminescent detection, the membrane wasstripped again and re-probed with an antibody against actin. See FIG.63.

In another experiment, lamina Cribrosa cell lines #506 and #517 weretransfected with 100 nM of siRNA. Six siRNAs were transfected, twocontrol siRNAs (siRNA #5 (SEQ ID NO:34) and one targeted against GAPDH)and four targeted against eIF-5A1 (siRNA #1 to #4) (SEQ ID NO:30-33).Seventy-two hours after transfection, the cell extract was harvested and5 μg of protein from each sample was separated on an SDS-PAGE gel,transferred to a PVDF membrane, and Western blotted with an antibodyagainst eIF-5A1. After chemiluminescent detection, the membrane wasstripped and re-probed with an antibody against actin. See FIG. 64.

In another experiment, the lamina cribrosa cell line #506 wastransfected with 100 nm of siRNA. Six siRNAs were transfected, twocontrol siRNAs (siRNA #5 (SEQ ID NO:34) and one targeted against GAPDH)and four targeted against eIF-5A1 (siRNA #1 to #4) (SEQ ID NO:30-33).Forty-eight hours after transfection, the media was replaced with mediacontaining 50 μM camptothecin and 10 ng/ml TNF-α. Twenty-four hourslater, the percentage of apoptotic cells was determined by labeling thecells with Hoescht. See FIG. 65.

In another experiment, the lamina cribrosa cell line #506 wastransfected with 100 nm of siRNA. Six siRNAs were transfected, twocontrol siRNAs (siRNA #5 (SEQ ID NO:34) and one targeted against GAPDH)and four targeted against eIF-5A1 (siRNA #1 to #4) (SEQ ID NO:30-33).Seventy-two hours after transfection, the media was replaced with mediacontaining 50 μM camptothecin and 10 ng/ml TNF-α. Twenty-four hourslater, the percentage of apoptotic cells was determined by labeling thecells with Hoescht. See FIG. 66.

In another experiment, the lamina cribrosa cell line #506 was eitherleft untransfected or was transfected with 100 nm of siRNA. Six siRNAswere transfected, two control siRNAs (siRNA #5 (SEQ ID NO:34) and onetargeted against GAPDH) and four targeted against eIF-5A1 (siRNA #1 to#4) (SEQ ID NO:30-33). Seventy-two hours after transfection, the mediawas replaced with media containing 50 μM camptothecin and 10 ng/mlTNF-α. Fresh media was also added to the untransfected, untreatedcontrol cells. Forty-eight hours later, the percentage of apoptoticcells was determined by labeling the cells with Hoescht. See FIG. 67.

Photographs of Hoescht-stained lamina cribrosa cell line #506transfected with siRNA and treated with camptothecin and TNF-α from theexperiment described in FIG. 67 and example 13. See FIG. 68.

Example 14

This example shows that treating a human cell line with antisenseoligonucleotides directed against apoptosis factor 5A causes the cellsto produce less TNF-α.

HepG2 cells were treated with 2.5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide eIF-5A1 #2 for a total oftwo days. Fresh media containing antisense oligonucleotides was addedafter 24 hours. Additional cells were left untreated for two days.Forty-eight hours after the beginning of treatment, the cells weretreated with IL-1β (1000 pg/ml) in fresh media for 6 hours. At the endof the experiment, the media was collected and frozen (−20° C.) forTNF-α quantification. TNF-α released into the media was measured usingELISA assays purchased from Assay Designs Inc. See FIG. 69.

Example 15

HT-29 cells (human colon adenocarcinoma) were transfected with either ansiRNA against eIF-5A1 or with a control siRNA with the reverse sequence.The siRNA used is as follows:

Position 690 (3'UTR) % G/C = 48 5′ AAGCUGGACUCCUCCUACACA 3′(SEQ ID NO: 79)The control siRNA used is as follows:

% G/C = 39 5′ AAACACAUCCUCCUCAGGUCG 3′ (SEQ ID NO: 80)After 48 hours the cells were treated with interferon-gamma (IFN-gamma)for 16 hours. After 16 hours the cells were washed with fresh media andtreated with lipopolysaccharide (LPS) for 8 or 24 hours. At each timepoint (8 or 24 hours) the cell culture media was removed from the cells,frozen, and the TNF-alpha present in the media was quantitated by ELISA.The cell lysate was also harvested, quantitated for protein, and used toadjust the TNF-alpha values to pg/mg protein (to adjust for differencesin cell number in different wells). The results of the Western blot andElisa are provided in FIGS. 74 A and B. FIG. 75 contains the results ofthe same experiment except the cells were at a higher density.

Example 16 Tissue Culture Conditions of U-937 Cell Line

U-937 is a human monocyte cell line that grows in suspension and willbecome adherent and differentiate into macrophages upon stimulation withPMA (ATCC Number CRL-1593.2) (cells not obtained directly from ATCC).Cells were maintained in RPMI 1640 media with 2 mM L-glutamine, 1.5 g/Lsodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvateand 10% fetal bovine serum in a 37° C. CO₂ (5%) incubator. Cells weresplit into fresh media (1:4 or 1:5 split ratio) twice a week and thecell density was always kept between 105 and 2×106 cells/ml. Cells werecultured in suspension in tissue culture-treated plastic T-25 flasks andexperiments were conducted in 24-well plates.

Time Course Experiment

Two days before the start of an experiment, the cell density wasadjusted to 3×105 cells/ml media. On the day of the experiment, thecells were harvested in log phase. The cell suspension was transferredto 15 ml tubes and centrifuged at 400×g for 10 mins at room temperature.The supernatant was aspirated and the cell pellet was washed/resuspendedwith fresh media. The cells were again centrifuged at 400×g for 10 mins,the supernatant was aspirated, and the cell pellet was finallyresuspended in fresh media. Equal volumes of cell suspension and trypanblue solution (0.4% trypan blue dye in PBS) were mixed and the livecells were counted using a haemocytometer and a microscope. The cellswere diluted to 4×105 cells/ml.

A 24-well plate was prepared by adding either PMA or DMSO (vehiclecontrol) to each well. 1 ml of cell suspension was added to each well sothat each well contained 400,000 cells, 0.1% DMSO+/−162 nM PMA. Thecells were maintained in a 37° C. CO₂ (5%) incubator. Separate wells ofcells were harvested at times 0, 24, 48, 72, 96, 99 and 102 h. See FIG.76 for a summary of the experimental time points and additions.

The media was changed at 72 h. Since some cells were adherent and otherswere in suspension, care was taken to avoid disrupting the adherentcells. The media from each well was carefully transferred intocorresponding microcentrifuge tubes and the tubes were centrifuged at14,000×g for 3 min. The tubes were aspirated, the cell pellets wereresuspended in fresh media (1 ml, (−) DMSO, (−) PMA), and returned totheir original wells. The cells become quiescent in this fresh mediawithout PMA. At 96 h, LPS (100 ng/ml) was added and cells were harvestedat 3 h (99 h) and 6 h (102 h) later.

At the time points, the suspension cells and media were transferred fromeach well into microcentrifuge tubes. The cells were pelleted at14,000×g for 3 min. The media (supernatant) was transferred to cleantubes and stored (−20° C.) for ELISA/cytokine analysis. The cellsremaining in the wells were washed with PBS (1 ml, 37° C.) and this PBSwas also used to wash the cell pellets in the correspondingmicrocentrifuge tubes. The cells were pelleted again at 14,000×g for 3min. The cells were lysed with boiling lysis buffer (50 mM Tris pH 7.4and 2% SDS). The adherent cells and the suspension cells from each wellwere pooled. The samples were boiled and then stored at −20° C.

Western Blotting

The protein concentration in each cell sample was determined by the BCA(bicinchoninic acid) method using BSA (bovine serum albumin) as thestandard protein. Protein samples (5 μg total protein) were separated by12% SDS-PAGE electrophoresis and transferred to PVDF membranes. Themembranes were blocked with polyvinyl alcohol (1 μg/ml, 30 sec) and with5% skim milk in PBS-t (1 h). The membranes were probed with a mousemonoclonal antibody raised against human eIF-5A (BD Biosciences cat#611976; 1:20,000 in 5% skim milk, 1 h). The membranes were washed 3×10mins PBS-t. The secondary antibody was a horseradishperoxidase-conjugated antimouse antibody (Sigma, 1:5000 in 1% skim milk,1 h). The membranes were washed 3×10 mins PBS-t. The protein bands werevisualized by chemiluminescence (ECL detection system, AmershamPharmacia Biotech).

To demonstrate that similar amounts of protein were loaded on each gellane, the membranes were stripped and reprobed for actin. Membranes werestripped (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7; 50°C. for 30 mins), washed, and then blocked as above. The membranes wereprobed with actin primary antibody (actin monoclonal antibody made inmouse; Oncogene, Ab-1; 1:20,000 in 5% skim milk). The secondaryantibody, washing, and detection were the same as above.

FIG. 77 shows that eIF-5A is upregulated during monocyte (U-397)differentiation and subsequent TNF-α secretion.

Example 17 Suppression of II-8 Production in Response to InterferonGamma by eIF-5A siRNA

HT-29 (human colon adenocarcinoma) cells were transfected with siRNAdirected to apoptosis eIF-5A. Approximately 48 hours after transfectionthe media was changed so that some of the test samples had media withinterferon gamma and some of the samples had media without interferongamma. 16 hours after interferon gamma addition, the cells were washed,and the media, with or without TNF-alpha, was placed on the cells. Themedia (used for ELISA detection of IL-8) and the cell lysate washarvested 8 or 24 hours later.

FIGS. 79 and 80 show that IL-8 is produced in response to TNF-alpha aswell as in response to interferon. Priming the cells with interferongamma prior to TNF treatment causes the cells to produce more IL-8 thaneither treatment alone. This may be due to the known upregulation of theTNF receptor 1 in response to interferon, so ‘priming’ the cells withinterferon allows them to respond to TNF better since the cells havemore receptors. siRNA against eIF-5A had no effect on IL-8 production inresponse to TNF alone (previous experiment) however, the siRNA blockedalmost all IL-8 produced in response to interferon as well as asignificant amount of the IL-8 produced as a result of the combinedtreatment of interferon and TNF. These results show that the by usingsiRNAs directed against apoptosis eIF-5A, the inventors have blocked theinterferon signaling pathway leading to IL-8, but not the TNF pathway.FIG. 81 is a western showing upregulation (4 fold at 8 hours) ofapoptosis eIF-5A in response to interferon gamma in HT-29 cells.

Example 18 Human Lamina Cribrosa Culture

Paired human eyes were obtained within 48 hours post mortem from the EyeBank of Canada, Ontario Division. Optic nerve heads (with attached pole)were removed and placed in Dulbecco's modified Eagle's medium (DMEM)supplemented with antibiotic/antimycotic, glutamine, and 10% FBS for 3hours. The optic nerve head (ONH) button was retrieved from each tissuesample and minced with fine dissecting scissors into four small pieces.Explants were cultured in 12.5 cm² plastic culture flasks in DMEMmedium. Growth was observed within one month in viable explants. Oncethe cells reached 90% confluence, they were trypsinized and subjected todifferential subculturing to produce lamina cribrosa (LC) and astrocytecell populations. LC cells were enriched by subculture in 25 cm² flasksin DMEM supplemented with gentamycin, glutamine, and 10% FBS. Cells weremaintained and subcultured as per this protocol.

The identity and population purity of cells populations obtained bydifferential subculturing was characterized using differentialfluorescent antibody staining on 8 well culture slides. Cells were fixedin 10% formalin solution and washed three times with Dulbecco'sPhosphate Buffered Saline (DPBS). Following blocking with 2% nonfat milkin DPBS, antibodies were diluted in 1% BSA in DPBS and applied to thecells in 6 of the wells. The remaining two wells were treated with only1% bovine serum albumin (BSA) solution and only secondary antibody ascontrols. Cells were incubated with the primary antibodies for one hourat room temperature and then washed three times with DPBS. Appropriatesecondary antibodies were diluted in 1% BSA in DPBS, added to each welland incubated for 1 hour. Following washing with DPBS, the slide waswashed in water, air-dried, and overlayed with Fluoromount (VectorLaboratories). Immunofluorescent staining was viewed under a fluorescentmicroscope with appropriate filters and compared to the control wellsthat were not treated with primary antibody. All primary antibodies wereobtained from Sigma unless otherwise stated. All secondary antibodieswere purchased from Molecular Probes. Primary antibodies used toidentify LC cells were: anti-collagen I, anti-collagen IV, anti-laminin,anti-cellular fibronectin, anti-glial fibrillary acidic protein (GFAP),and anti-alpha-smooth muscle actin. Cell populations were determined tobe comprised of LC cells if they stained positively for collagen I,collagen to IV, laminin, cellular fibronectin, alpha smooth muscle actinand negatively for glial fibrillary (GFAP). In this study, two sets ofhuman eyes were used to initiate cultures. LC cell lines #506 and #517were established from the optic nerve heads of and 83-year old male anda 17-year old male, respectively. All LC cell lines have been fullycharacterized and found to contain greater than 90% LC cells.

Treatment of LC Cells

Apoptosis was induced in lamina cribrosa cells using a combination of 50μM camptothecin (Sigma) and 10 ng/ml TNF-α (Leinco Technologies). Thecombination of camptothecin and TNF-α was found to be more effective atinducing apoptosis than either camptothecin or TNF-α alone.

Construction and Transfection of siRNAs

Small inhibitory RNAs (siRNAs) directed against human eIF-5A were usedto specifically suppress expression of eIF-5A in lamina cribrosa cells.Six siRNAs were generated by in vitro transcription using the Silencer™siRNA Construction Kit (Ambion Inc.). Four siRNAs were generated againsthuman eIF-5A1 (siRNAs #1 to #4). Two siRNAs were used as controls; ansiRNA directed against GAPDH provided in the kit, and an siRNA (siRNA#5), which had the reverse sequence of the eIF-5A-specific siRNA #1, butdoes not itself target eIF-5A. The siRNAs were generated according tothe manufacturer's protocol. The eIF-5A and control siRNA targets hadthe following sequences: siRNA #1 5′ AAAGGAATGACTTCCAGCTGA 3′ (SEQ IDNO: 81); siRNA #2 5′ AAGATCGTCGAGATGTCTACT 3′ (SEQ ID NO: 82); siRNA #35′ AAGGTCCATCTGGTTGGTATT 3′ (SEQ ID NO: 83); siRNA #4 5′AAGCTGGACTCCTCCTACACA 3′ (SEQ ID NO: 84); siRNA #5′AAAGTCGACCTTCAGTAAGGA 3′ (SEQ ID NO: 85). Lamina cribrosa cells weretransfected with siRNA using LipofectAMINE 2000. Lamina cribrosa cellswere transfected when cell confluence was at 40 to 70% and weregenerally seeded onto 8-well culture slides at 7500 cells per well threedays prior to transfection. Transfection medium sufficient for one wellof an 8-well culture slide was prepared by diluting 25.5 pmoles of siRNAto a final volume of 21.2 μl in Opti-Mem (Sigma). 0.425 μl ofLipofectamine 2000 was diluted to a final volume of 21.2 μl in Opti-Memand incubated for 7 to 10 minutes at room temperature. The dilutedLipofectamine 2000 mixture was then added to the diluted siRNA mixtureand incubated together at room temperature for 20 to 30 minutes. Thecells were washed once with serum-free media before adding 135 μl ofserum-free media to the cells and overlaying 42.4 μl of transfectionmedium. The cells were placed back in the growth chamber for 4 hours.After the incubation, 65 μl of serum-free media plus 30% FBS was addedto the cells. Transfection of siRNA into cells to be used for Westernblot analysis were performed in 24-well plates using the same conditionsas the transfections in 8-well slides except that the volumes wereincreased by 2.3 fold. Following transfection, lamina cribrosa cellswere incubated for 72 hours prior to treatment with 50 μM ofcamptothecin (Sigma) and 10 ng/ml of TNF-α (Leinco Technologies) toinduce apoptosis. Cell lysates were then harvested for Western blottingor the cells were examined for apoptosis

Detection of Apoptotic Cells

Transfected cells that had been treated with TNF-α and camptothecin for24 hours were stained with Hoescht 33258 in order to determine thepercentage of cells undergoing apoptosis. Briefly, cells were fixed witha 3:1 mixture of absolute methanol and glacial acetic acid and thenincubated with Hoescht stain (0.5 μg/ml Hoescht 33258 in PBS). After a10 minute incubation in the dark, the staining solution was discarded,the chambers separating the wells of the culture slide were removed, andthe slide was washed 3 times for 1 minute with deionized water. Afterwashing, a few drops of McIlvaine's buffer (0.021 M citric acid, 0.058 MNa₂HPO₄.7H₂O; pH 5.6) was added to the cells and overlaid with acoverslip. The stained cells were viewed under a fluorescent microscopeusing a UV filter. Cells with brightly stained or fragmented nuclei werescored as apoptotic. A minimum of 200 cells were counted per well. TheDeadEnd™ Fluorometric TUNEL (Promega) was also used to detect the DNAfragmentation that is a characteristic feature of apoptotic cells.Following Hoescht staining, the culture slide was washed briefly withdistilled water, and further washed by immersing the slide twice for 5minutes in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mMNa₂HPO₄), blotting the slide on paper towel between washes. The cellswere permeabilized by immersing them in 0.2% Triton X-100 in PBS for 5minutes. The cells were then washed again by immersing the slide twicefor 5 minutes in PBS and blotting the slide on paper towel betweenwashes. 25 μl of equilibration buffer [200 mM potassium cacodylate (pH6.6), 25 mM Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg/ml bovineserum albumin, and 2.5 mM cobalt chloride] was added per well andincubated for 5 to 10 minutes. During equilibration, 30 μl of reactionmixture was prepared for each well by mixing in a ratio of 45:5:1,respectively, equilibration buffer, nucleotide mix [50 μMfluorescein-12-dUTP, 100 μM dATP, 10 mM Tris-HCl (pH 7.6), and 1 mMEDTA], and terminal deoxynucleotidyl transferase enzyme (Tdt, 25 U/μl).After the incubation in equilibration buffer, 30 μl of reaction mixturewas added per well and overlayed with a coverslip. The reaction wasallowed to proceed in the dark at 37° C. for 1 hour. The reaction wasterminated by immersing the slide in 2×SSC [0.3 M NaCl, and 30 mM sodiumcitrate (pH 7.0)] and incubating for 15 minutes. The slide was thenwashed by immersion in PBS three times for 5 minutes. The PBS wasremoved by sponging around the wells with a Kim wipe, a drop of mountingmedia (Oncogene research project, JA1750-4ML) was added to each well,and the slide was overlayed with a coverslip. The cells were viewedunder a fluorescent microscope using a UV filter (UV-G 365, filter set487902) in order to count the Hoescht-stained nuclei. Any cells withbrightly stained or fragmented nuclei were scored as apoptotic. Usingthe same field of view, the cells were then viewed using a fluoresceinfilter (Green H546, filter set 48915) and any nuclei fluorescing brightgreen were scored as apoptotic. The percentage of apoptotic cells in thefield of view was calculated by dividing the number of bright greennuclei counted using the fluorescein filter by the total number ofnuclei counted under the UV filter. A minimum of 200 cells were countedper well.

Protein Extraction and Western Blot Analysis

Protein was isolated for Western blotting from lamina cribrosa cellsgrowing on 24-well plates by washing the cells twice in PBS (8 g/L NaCl,0.2 g/L KCl, 1.44 g/L Na₂HPO₄, and 0.24 g/L KH₂PO₄) and then adding 50μl of lysis buffer [2% SDS, 50 mM Tris-HCl (pH 7.4)]. The cell lysatewas collected in a microcentrifuge tube, boiled for 5 minutes and storedat −20° C. until ready for use. Protein concentrations were determinedusing the Bicinchoninic Acid Kit (BCA; Sigma). For Western blotting, 5μg of total protein was separated on a 12% SDS-polyacrylamide gel. Theseparated proteins were transferred to a polyvinylidene difluoridemembrane. The membrane was then incubated for one hour in blockingsolution (5% skim milk powder, 0.02% sodium azide in PBS) and washedthree times for 15 minutes in PBS-T (PBS+0.05% Tween-20). The membranewas stored overnight in PBS-T at 4° C. After being warmed to roomtemperature the next day, the membrane was blocked for 30 seconds in 1μg/ml polyvinyl alcohol. The membrane was rinsed 5 times in deionizedwater and then blocked for 30 minutes in a solution of 5% milk in PBS.The primary antibody was preincubated for 30 minutes in a solution of 5%milk in PBS prior to incubation with the membrane. The primaryantibodies used were anti-eIF-5A (BD Transduction Laboratories) at1:20,000 and anti-β-actin (Oncogene). The membranes were washed threetimes in PBS-T and incubated for 1 hour with the appropriateHRP-conjugated secondary antibodies diluted in 1% milk in PBS. The blotwas washed and the ECL Plus Western blotting detection kit (AmershamPharmacia Biotech) was used to detect the peroxidase-conjugated boundantibodies.

Results

Two lamina cribrosa (LC) cell lines were established from optic nerveheads obtained from male donors ranging in age from 83 years (#506) to17 years (#517). The cells isolated from the human lamina cribrosa hadthe same broad, flat morphology with prominent nucleus observed in otherstudies (Lambert et al., 2001). Consistent with the characterizations ofother groups, the LC cells showed immunoreactivity to alpha smoothmuscle actin (FIG. 82 a) as well as to a number of extracellular matrixproteins including cellular fibronectin (FIG. 82 b), laminin (FIG. 82c), collagen I, and collagen IV (data not shown) (Clark et al., 1995;Hernandez et al., 1998; Hernandez and Yang, 2000; Lambert et al.; 2001).Negative immunoreactivity of the LC cells to glial fibrillary acidicprotein (GFAP) was also observed consistent with previous findings (FIG.82 d) (Lambert et al., 2001). These findings support the identificationof the isolated cells as being LC cells rather than optic nerve headastrocytes.

Since TNF-α is believed to play an important role during theglaucomatous process, the susceptibility of LC cells to the cytotoxiceffects of TNF-α was examined. Confluent LC cells were exposed to eithercamptothecin, TNF-α, or a combination of camptothecin and TNF-α for 48hours (FIG. 83). Hoescht staining revealed that TNF-α alone was notcytotoxic to LC cells. Treatment with camptothecin resulted inapproximately 30% cell death of the LC cells. However, a synergisticincrease in apoptosis was observed when LC cells were treated with bothcamptothecin and TNF-α, a treatment which resulted in the death of 45%of LC cells by 48 hours. These results indicate that LC cells arecapable of responding to the cytotoxic effects of TNF-α when primed forapoptosis by camptothecin.

EIF-5A is a nucleocytoplasmic shuttle protein known to be necessary forcell division and recently suggested to also be involved duringapoptosis. We examined the expression of eIF-5A protein in LC cellsbeing induced to undergo apoptosis by either camptothecin, orcamptothecin plus TNF-α. The expression of eIF-5A was not alteredsignificantly upon treatment with camptothecin except perhaps todecrease slightly (FIG. 84A). However, a significant upregulation ofeIF-5A protein was observed after 8 and 24 hours of camptothecin plusTNF-α treatment (FIG. 84B). These results indicate that eIF-5Aexpression is induced specifically by exposure to TNF-α and expressioncorrelates to the induction of apoptosis. This points to a role foreIF-5A in the apoptotic pathway downstream of TNF-α receptor binding.

In order to examine the importance of eIF-5A expression duringTNF-α-induced apoptosis in LC cells, a series of four siRNAs (siRNAs #1to #4) targeting eIF-5A were designed and synthesized by in vitrotranscription. To determine the effectiveness of the siRNAs insuppressing eIF-5A protein expression, LC cell lines #506 and #517 weretransfected with each of the siRNAs and expression of eIF-5A protein inthe cell lysate was examined 72 hours later (FIG. 85). For comparison,cells were also transfected with either an siRNA against GAPDH and/or acontrol siRNA (siRNA #5) having the same chemical composition as siRNA#1 but which does not recognize eIF-5A. All siRNAs directed againsteIF-5A were capable of significantly suppressing eIF-5A expression inboth LC cell lines (FIG. 85). The GAPDH siRNA was used as an additionalcontrol because, unlike the control siRNA #5 which simply has thereverse sequence of siRNA #1 and does not have a cellular target, it isan active siRNA capable of suppressing the expression of its targetprotein, GAPDH (data not shown). All four siRNAs against eIF-5A werealso capable of protecting transfected LC cells (#506) from apoptosisinduced by 24 hour treatment with TNF-α and camptothecin (FIG. 86).Using Hoescht staining to detect cell death, the siRNAs (siRNAs #1 to#4) were found to be able to reduce apoptosis of LC cells by 59% (siRNA#1), 35% (siRNA #2), 50% (siRNA #3), and 69% (siRNA #4). Interestingly,the siRNA against GAPDH was also able to reduce apoptosis of LC cells by42% (FIG. 86). GAPDH is known to have cellular functions outside of itsrole as a glycolytic enzyme, including a proposed function duringapoptosis of cerebellar neurons (Ishitani and Chuang, 1996; Ishitani etal., 1996a; Ishitani et al., 1996b). In a similar experiment we alsodemonstrated that siRNA #1 was able to reduce apoptosis of the LC line#517 by 53% in response to TNF-α and camptothecin indicating that eIF-5AsiRNAs are protective for LC cells isolated from different optic nerveheads (FIG. 87). These results indicate that eIF-5A does have a functionduring apoptosis and may be an important intermediate in the pathwayleading to TNF-α-induced apoptosis in LC cells.

In order to confirm that LC cells exposed to TNF-α and camptothecin weredying by classical apoptosis, DNA fragmentation was evaluated in situusing the terminal deoxynucleotidyl transferase-mediateddUTP-digoxigenin nick end labeling (TUNEL) method. LC cells (#506) weretreated with TNF-α and camptothecin for 24 hours, 3 days aftertransfection with either an eIF-5A siRNA (siRNA #1) or a control siRNA(siRNA #5). The cells were also stained with Hoescht to facilitatevisualization of the nuclei. 46% of LC cells transfected with thecontrol siRNA were positive for TUNEL staining while only 8% of LC cellstransfected with eIF-5A siRNA #1 were positively labeled indicating thatthe eIF-5A siRNA provided greater than 80% protection from apoptosis(FIG. 88). Similar results were obtained with eIF-5A siRNA #4 whichprovided greater than 60% protection from apoptosis relative to thecontrol siRNA (data not shown).

1. An siRNA for suppressing expression of apoptosis-specific eIF-5A1wherein the siRNA targets SEQ ID NO:53 and wherein the siRNA isdouble-stranded for 19-25 nucleotides in length.
 2. The siRNA of claim1, wherein the siRNA has at least one single-stranded overhang region,with each single-stranded region comprising six or fewer nucleotides. 3.The siRNA of claim 2, wherein the siRNA has two single-stranded overhangregions.
 4. The siRNA of claim 3, wherein each of the twosingle-stranded overhang regions comprise two nucleotides or less. 5.The siRNA of claim 4, wherein the double-stranded region on the siRNA is19-21 nucleotides in length.
 6. The siRNA of claim 5, wherein thedouble-stranded region on the siRNA is 21 nucleotides in length.
 7. ThesiRNA of claim 6, wherein one strand of the siRNA has the nucleotidesequence of SEQ ID NO:54.
 8. The siRNA of claim 6, wherein one strand ofthe siRNA has the nucleotide sequence of SEQ ID NO:55.
 9. The siRNA ofclaim 5, wherein the double-stranded region on the siRNA is 19nucleotides in length.
 10. The siRNA of claim 9, wherein one strand ofthe siRNA has the nucleotide sequence of 5′-GCUGGACUCCUCCUACACA-3′ (SEQID NO:86).
 11. The siRNA of claim 9, wherein one strand of the siRNA hasthe nucleotide sequence of 5′-UGUGUAGGAGGAGUCCAGC-3′ (SEQ ID NO:87).